EP1750699A2 - Traitement de conditions relatives au régime utilisant des inhibieursphospholipase-a2 comprenant des indoles et des composés relatifs - Google Patents

Traitement de conditions relatives au régime utilisant des inhibieursphospholipase-a2 comprenant des indoles et des composés relatifs

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Publication number
EP1750699A2
EP1750699A2 EP05779544A EP05779544A EP1750699A2 EP 1750699 A2 EP1750699 A2 EP 1750699A2 EP 05779544 A EP05779544 A EP 05779544A EP 05779544 A EP05779544 A EP 05779544A EP 1750699 A2 EP1750699 A2 EP 1750699A2
Authority
EP
European Patent Office
Prior art keywords
phospholipase
inhibitor
ofthe
group
cholesterol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05779544A
Other languages
German (de)
English (en)
Other versions
EP1750699A4 (fr
Inventor
Jerry M. Buysse
Dominique Charmot
Han-Ting Chang
Michael James Cope
David Hui
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ilypsa Inc
Original Assignee
Ilypsa Inc
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Publication date
Application filed by Ilypsa Inc filed Critical Ilypsa Inc
Publication of EP1750699A2 publication Critical patent/EP1750699A2/fr
Publication of EP1750699A4 publication Critical patent/EP1750699A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Phospholipases are a group of enzymes that play important roles in a number of biochemical processes, including regulation of membrane fluidity and stability, digestion and metabolism of phospholipids, and production of intracellular messengers involved in inflammatory pathways, hemodynamic regulation and other cellular processes. Phospholipases are themselves regulated by a number of mechanisms, including selective phosphorylation, pH, and intracellular calcium levels. Phospholipase activities can be modulated to regulate their related biochemical processes, and a number of phospholipase inhibitors have been developed. [0003] A large number of phospholipase- A2 (PL A2 or PL A 2 ) inhibitors are known in the art.
  • PL A 2 inhibiting moieties include, for example, small molecule inhibitors as well as phospholipid analog and transition state analog compounds. Many such small-molecule inhibitors were developed, for example, for indications related to inflammatory states.
  • a non-exhaustive, exemplification of known phospholipase- A2 inhibitors include the following classes: Alkynoylbenzoic, -Thiophenecarboxylic, - Furancarboxylic, and -Pyridinecarboxylic acids (e.g. see US5086067); Amide carboxylate derivatives (e.g. see WO9108737); Aminoacid esters and amide derivatives (e.g. see WO2002008189); Aminotetrazoles (e.g.
  • Cinnamic acid derivatives e.g. see US5578639
  • Cyclohepta-indoles e.g. see WO03016277
  • Ethaneamine-benzenes Imidazolidinones, Thiazoldinones and Pyrrolidmones (e.g. see WO03031414);
  • lndole glyoxamides e.g. see US5654326); Lidole glyoxamides (e.g. see WO9956752); Indoles (e.g. see US6630496 and WO9943672; hidoly (e.g.
  • WO003048122 hidoly containing sulfonamides; N-cyl-N-cinnamoylethylenediamine derivatives (e.g. see WO9603371); Naphyl acateamides (e.g. see EP77927); N- substituted glycines (e.g. see US 5298652); Phosopholipid analogs (e.g. see US5144045 and US6495596); Piperazines (e.g. see O03048139); Pyridones and Pyrimidones (e.g. see WO03086400); 6-carbamoylpicolinic acid derivatives (e.g.
  • Pancreatic phospholipase A2 IB (PLA2 LB) is thought to play a role in phospholipid digestion and processing.
  • PLA2 IB is an enzyme having activity for catabolizing phosphatidylcholine (PC) to form lysophosphatidylcholine (LPC) and free fatty acid (FFA) as reaction products.
  • PC phosphatidylcholine
  • LPC lysophosphatidylcholine
  • FFA free fatty acid
  • PLA2 deficient mice PLA2 (-/-) mice, also referred to herein as PLA2 knock-out mice
  • PLA2 (-/-) mice PLA2 knock-out mice
  • the PLA2 (-/-) mice were fed with a normal chow, indicated that the cholesterol abso ⁇ tion efficiency and the plasma lipid level were similar to the wild-type mice PLA2 (+/+).
  • Creaseau, et al. "Compensatory phospholipid digestion is required for cholesterol abso ⁇ tion in pancreatic phospholipase A(2)-deficient mice.”
  • Gastroenterology 120(5): 1193-202 The same study also showed that in the PLA2 (-/-) group, intestinal PC was fully hydrolyzed even in the absence of pancreatic PLA2 activity.
  • the PLA2 (-/-) mice on high-fat / high-cholesterol diet were reported to have: reduced body weight gain over a sixteen week period, with the observed weight difference being due to increased adiposity in the wild-type mice; substantially lower fasting plasma leptin concentrations; improved glucose tolerance; and improved protection against high-fat-diet induced insulin resistance.
  • the effect did not produce overt steatorrhea, suggesting only a slight reduction in fat abso ⁇ tion.
  • Diabetes affects 18.2 million people in the Unites States, representing over 6% of the population. Diabetes is characterized by the inability to produce or properly use insulin. Diabetes type 2 (also called non-insulin-dependent diabetes or NTDDM) accounts for 80-90% ofthe diagnosed cases of diabetes and is caused by insulin resistance. Insulin resistance in diabetes type 2 prevents maintenance of blood glucose within desirable ranges, despite normal to elevated plasma levels of insulin.
  • Obesity is a major contributor to diabetes type 2, as well as other illnesses including coronary heart disease, osteoarthritis, respiratory problems, and certain cancers. Despite attempts to control weight gain, obesity remains a serious health concern in the United States and other industrialized countries. Indeed, over 60% of adults in the United States are considered overweight, with about 22% of these being classified as obese.
  • lipid-related disorders include hypercholesterolemia and hypertriglyceridemia among other indications.
  • Non-HDL cholesterol is firmly associated with atherogenesis and its sequalea including cardiovascular diseases such as arteriosclerosis, coronary artery disease myocardial infarction, ischemic stroke, and other forms of heart disease. These together rank as the most prevalent type of illness in industrialized countries. Indeed, an estimated 12 million people in the United States suffer with coronary artery disease and about 36 million require treatment for elevated cholesterol levels.
  • HMG-CoA reductase inhibitors are reported to be used to reduce serum LDL cholesterol levels.
  • statins Hydroxymethylglutaryl-coenzym A reductase inhibitors
  • severe and sometimes fatal adverse events, including liver failure and rhabdomyolysis have been reported in connection with such use of statins.
  • ezitimibe was introduced as a cholesterol abso ⁇ tion inhibitor, for use alone or in combination with statins.
  • fibrates e.g. gemfibrozil
  • fibrates are used to lower high serum triglyceride concentrations.
  • the present invention provides methods, compositions, medicaments, foodstuffs and kits comprising phospholipase inhibitors having beneficial impact for treatment of phospholipase-related conditions, such as insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity) and/or cholesterol-related conditions.
  • phospholipase inhibitors having beneficial impact for treatment of phospholipase-related conditions, such as insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity) and/or cholesterol-related conditions.
  • One first aspect ofthe present invention relates to methods of treating one or more conditions such as diet-related conditions, including for example conditions selected from the group consisting of a weight-related condition, an insulin- related condition, a cholesterol-related condition and combinations thereof (preferably, including for example conditions selected from obesity, diabetes mellitus (e.g., diabetes type 2), insulin resistance, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and combinations thereof).
  • This method comprises, in one approach, administering an effective amount of a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof).
  • This method comprises, in another approach, administering an effective amount of a phospholipase- A 2 inhibitor (preferably a phospholipase- A 2 IB inhibitor) to a subject, the phospholipase- A 2 inhibitor comprising a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically- acceptable salt thereof).
  • a phospholipase- A 2 inhibitor preferably a phospholipase- A 2 IB inhibitor
  • Another second aspect ofthe invention is directed to methods for modulating the metabolism of fat, glucose or cholesterol (or combinations thereof) in a subject.
  • This method comprises, in one approach, administering an effective amount of a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof).
  • the method comprises administering an effective amount of a phospholipase- A 2 inhibitor
  • the phospholipase- A 2 inhibitor comprising a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof).
  • the invention in a third aspect, in one approach, relates to methods comprising use of a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof) for manufacture of a medicament for use as a pharmaceutical for treating a condition of a subject selected from a weight-related condition, an insulin-related condition, a cholesterol-related condition and combinations thereof (preferably, including for example conditions selected from obesity, diabetes mellitus, insulin resistance, glucose intolerance, hypercholesterolemia, hypertriglyceridemia and combinations thereof)
  • a phospholipase- A inhibitor preferably a phospholipase- A 2 LB inhibitor
  • the invention relates to methods comprising use of a phospholipase- A inhibitor (preferably a phospholipase- A 2 LB inhibitor) for manufacture of a medicament for use as a pharmaceutical for treating a condition of a subject selected from a weight-related condition, an insulin-related condition, a cholesterol-related condition and
  • the invention in a fourth aspect, in one approach, relates to a food product composition comprising an edible foodstuff and a substituted organic compound having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof).
  • the invention in another approach, relates to a food product composition comprising an edible foodstuff and a phospholipase- A 2 inhibitor (preferably a phospholipase- A IB inhibitor), the phospholipase- A 2 inhibitor comprising a substituted organic compound having a fused five-member ring and six- member ring (or as a pharmaceutically-acceptable salt thereof).
  • the foodstuff can comprise (or can consist essentially of) a vitamin supplement and the phospholipase- A2 inhibitor.
  • the phospholipase- A2 inhibitor can comprise a substituted organic compound (or including a moiety thereof) comprising a fused five-member ring and six-member ring having one or more heteroatoms (e.g., nitrogen, oxygen) substituted within the ring structure ofthe five-member ring, within the ring structure ofthe six-member ring, or within the ring structure of each ofthe five-member and six-member rings, and in each case with substituent groups effective for imparting phospholipase- A2 inhibiting functionality to the compound (or moiety).
  • a substituted organic compound or including a moiety thereof
  • the phospholipase- A2 inhibitor can comprise a substituted organic compound (or including a moiety thereof) comprising a fused five-member ring and six-member ring having one or more heteroatoms (e.g., nitrogen, oxygen) substituted within the ring structure ofthe five-member ring, within the ring structure ofthe six-member ring, or within the ring structure
  • a phospholipase- A2 inhibitor or inhibiting moiety can comprise an indole-containing moiety (referred to herein interchangeably as an indole or an indole compound or an indole-moiety), such as a substituted indole moiety.
  • an indole-containing moiety referred to herein interchangeably as an indole or an indole compound or an indole-moiety
  • Particularly-preferred indole compounds and moieties are disclosed further herein.
  • the phospholipase- A2 inhibitor can have lumen-localization functionality.
  • the phospholipase- A2 inhibitor can have chemical and physical properties that impart lumen-localization functionality to the inhibitor.
  • the inhibitors of these embodiments can have chemical and/or physical properties such that at least about 80% ofthe phospholipase inhibitor remains in the gastrointestinal lumen, and preferably at least about 90% ofthe phospholipase inhibitor remains in the gastrointestinal lumen (in each case, following administration ofthe inhibitor to the subject).
  • the phospholipase- A2 inhibitor can comprise or consist essentially ofthe substituted organic compound having a fused five-member ring and six-member ring.
  • the phospholipase inhibitor can comprise a moiety ofthe substituted organic compound having a fused five-member ring and six-member ring, with the moiety being linked (e.g., covalently linked, directly or indirectly using a linking moiety) to a non-absorbed or non-absorbable moiety, preferably to a non- absorbed or non-absorbable oligomer or polymer moiety.
  • the phospholipase- A 2 inhibitor does not induce substantial steatorrhea following administration or ingestion thereof.
  • these embodiments can be used in various and specific combination, and in each permutation, with other aspects and embodiments described above or below herein.
  • FIG. 1 is a schematic representation of a chemical reaction in which phospholipase- A2 enzyme (PLA2) catalyzes hydrolysis of phospholipids to corresponding lysophospholipids.
  • FIG. 2 is a chemical formula for [2-(3-(2-amino-2-oxoacetyl)-l-
  • FIG. 3 is a graph illustrating the results of Example 5 A, showing body weight gain in groups of mice receiving ILY-4001 at low dose (4001-L) and high dose (4001-H) as compared to wild-type control group (Control) and as compared to genetically deficient PLA2 (-/-) knock-out mice (PLA2 KO).
  • FIG. 4 is a graph illustrating the results of Example 5B, showing fasting serum glucose levels in groups of mice receiving ILY-4001 at low dose (4001-L) and high dose (4001-H) as compared to wild-type control group (Control) and as compared to genetically deficient PLA2 (-/-) knock-out mice (PLA2 KO).
  • FIG.'s 5A and 5B are graphs illustrating the results of Example 5C, showing serum cholesterol levels (Fig. 5A) and serum triglyceride levels (Fig. 5B) in groups of mice receiving ILY-4001 at low dose (4001-L) and high dose (4001-H) as compared to wild-type control group (Control) and as compared to genetically deficient PLA2 (-/-) knock-out mice (PLA2 KO).
  • FIG.'s 6 A through 6D are schematic representations including chemical formulas illustrating indole compounds (Fig. 6A, Fig. 6C and Fig. 6D) and indole- related compounds (Fig. 6B).
  • FIG.'s 7 A and 7B are a schematic representation (Fig. 7 A) of an in-vitro fluorometric assay for evaluating PLA2 IB enzyme inhibition, and a graph (Fig. 7 A).
  • FIG. 7B shows the results of Example 6 A in which the assay was used to evaluate ILY-4001 [2-(3 -(2-amino-2-oxoacetyl)- 1 -(biphenyl-2-ylmethyl)-2-methyl- 1 H-indol-4- yloxy)acetic acid].
  • FIG.'s 8 A and 8B are graphs showing the results from the in-vitro Caco- 2 permeability study of Example 6B for ILY-4001 [2-(3 -(2-amino-2-oxoacetyl)- 1 - (biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] (Fig.
  • FIG. 9 is a schematic illustration, including chemical formulas, which outlines the overall synthesis scheme for ILY-4001 [2-(3-(2-amino-2-oxoacetyl)-l- (bi ⁇ henyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] as described in Example 4.
  • ILY-4001 2-(3-(2-amino-2-oxoacetyl)-l- (bi ⁇ henyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid
  • the present invention provides phospholipase inhibitors, compositions
  • the phospholipase inhibitors ofthe present invention can find use in treating a number of phospholipase-related conditions, including insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity), cholesterol-related disorders and any combination thereof, as described in detail below.
  • insulin-related conditions e.g., diabetes
  • weight-related conditions e.g., obesity
  • cholesterol-related disorders e.g., cholesterol-related disorders and any combination thereof, as described in detail below.
  • the invention comprises, in one aspect, a method of treating such conditions by administering an effective amount of a phospholipase- A 2 IB inhibitor to a subject, where the phospholipase- A 2 IB inhibitor comprises a substituted organic compound having a fused five-member ring and six-member ring.
  • the invention also contemplates, in another aspect, a method for modulating the metabolism of fat, glucose or cholesterol in a subject by administering an effective amount of such phospholipase- A 2 IB inhibitor to the subject.
  • the invention includes as well, in a further aspect, methods of using a phospholipase- A 2 IB inhibitor for manufacture of a medicament, where the medicament is indicated for use as a pharmaceutical for treating a condition of a subject (e.g., a weight-related condition, an insulin-related condition, a cholesterol- related condition and combinations thereof), and where the phospholipase- A 2 IB inhibitor comprises a substituted organic compound having a fused five-member ring and six-member ring.
  • a condition of a subject e.g., a weight-related condition, an insulin-related condition, a cholesterol- related condition and combinations thereof
  • the invention can include, moreover in another aspect, a food product composition comprising an edible foodstuff and a phospholipase- A IB inhibitor, preferably where the phospholipase- A 2 IB inhibitor comprises the substituted organic compound having a fused five-member ring and six-member ring.
  • the phospholipase inhibitor (or inhibiting moiety) can comprise a substituted organic compound (or moiety derived from a substituted organic compound) having a fused five-member ring and six-member ring (or as a pharmaceutically-acceptable salt thereof).
  • the inhibitor also comprises substituent groups effective for imparting phospholipase- A2 inhibiting functionality to the inhibitor (or inhibiting moiety), and preferably phospholipase- A2 IB inhibiting functionality.
  • the phospholipase inhibitor a fused five-member ring and six-member ring having one or more heteroatoms (e.g., nitrogen, oxygen, sulfer) substituted within the ring structure of the five-member ring, within the ring structure ofthe six-member ring, or within the ring structure of each ofthe five-member and six-member rings (or as a pharmaceutically-acceptable salt thereof).
  • the inhibitor (or inhibiting moiety) can comprise substituent groups effective for imparting phospholipase inhibiting functionality to the moiety.
  • substituted organic compounds (or moieties derived therefrom) having such fused five-member ring and six-member ring are effective phospholipase-2A IB inhibitors, with phenotypic effects approaching and/or comparable to the effect of genetically deficient PLA2 (-/-) mice.
  • such compound (or moieties derived therefrom) are effective in treating conditions such as weight-related conditions, insulin-related conditions, and cholesterol-related conditions, including in particular conditions such as obesity, diabetes mellitus, insulin resistance, glucose intolerance, hypercholesterolemia and hypertriglyceridemia.
  • compounds comprising the fused five-membered and six- membered rings have a structure that advantageously provides an appropriate bond- length and bond-angles for positioning substituent groups - for example at positions 3 and 4 of an indole-compound as represented in Figure 6A, and at the -R 3 and -R positions ofthe indole-related compounds comprising fused five-membered and six- membered rings as represented in Figure 6B.
  • Minor-image analogues of such indole compounds and of such indole-related compounds also can be used in connection with this invention, as described below.
  • the phospholipase- A2 inhibitor can comprise indole compounds or indole-related compounds.
  • the phospholipase- A2 inhibitor (or inhibiting moiety) can be a lumen-localized phospholipase-A2 inhibitor.
  • the phospholipase- A2 inhibiting moiety can comprise a fused five-membered ring and six-membered ring as a compound (or as a pharmaceutically-acceptable salt thereof), represented by the following formula (I):
  • R ⁇ through R 7 are independently selected from the group consisting of: hydrogen, oxygen, sulfur, phosphorus, amine, halide, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, acylamino, oxi yl, hydrazyl, substituted substitution group, and combinations thereof; and additionally or alternatively, wherein R t through R 7 can optionally comprise, independently selected additional rings between two adjacent substitutents, with such additional rings being independently selected 5-, 6-, and/or 7-member rings which are carbocyclic rings, heterocyclic rings, and combinations thereof.
  • an amine group can include primary, secondary and tertiary amines; a halide group can include fluoro, chloro, bro o, or iodo; a carbonyl group can be a carbonyl moiety having a further substitution (defined below) as represented by the formula
  • substitution an acidic group can be an organic group as a proton donor and capable of hydrogen bonding, non-limiting examples of which include carboxylic acid, sulfate, sulfonate, phosphonates, substituted phosphonates, phosphates, substituted phosphates, 5-tetrazolyl,
  • an alkyl group by itself or as part of another substituent can be a substituted or unsubstituted straight or branched chain hydrocarbon such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tertiary butyl, sec-butyl, n-pentyl, n-hexyl, decyl, dodecyl, or octadecyl;
  • an alkenyl group by itself or in combination with other group can be a substituted or unsubstituted straight chain or branched hydrocarbon containing unsaturated bonds such as vinyl, propenyl, crotonyl, isopentenyl, and various butenyl isomers;
  • a carbocyclic group can be a substituted or unsubstituted, saturated or unsaturated, 5- to 14-membered organic nucleus whose ring forming atoms are solely carbon atoms, including cycl
  • an oximyl group can be an oximyl moiety having two further substitutions (defined below) as represented by the formula:
  • a hydrazyl group can be a hydrazyl moiety having three three further substitutions (defined below) as represented by the formula: urther substitution
  • a substituted substitution group combines one or more ofthe listed substituent groups, preferably through moieties that include for example an — oxygene — alkyl — acidic moiety such as .C0 2 H carbonyl — acyl amino — hydrogen moiety such as an — alkyl — carbocyclic — alkenyl moiety such as
  • a further substitution group can mean a group selected from hydrogen, oxygen, sulfur, phosphorus, amine, halide, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group, and combinations thereof.
  • the phospholipase- A2 inhibiting moiety can comprise an indole compound (e.g., an indole-containing compound or compound containing an indole moiety), such as a substituted indole moiety.
  • an indole compound e.g., an indole-containing compound or compound containing an indole moiety
  • the indole-containing compound can be a compound represented by the formulas II, III (considered left to right as shown):
  • K ⁇ through R 7 are independently selected from the groups consisting of: hydrogen, oxygen, sulfur, phosphorus, amine, halide, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group, and combinations thereof; and additionally or alternatively, wherein R 1 through R can optionally, and independently form additional rings between two adjacent substitutents with such additional rings being 5-, 6-, and 7- member ring selected from the group consistin of carbocyclic rings, heterocyclic rings and combinations thereof.
  • Some indole compounds having additional rings include, for example, those compounds represented as formulas IVa through IVf (considered left to right in top row as IVa, IVb, IVc, and considered left to right bottom row as IVd, IVe and IVf, as shown):
  • substituent groups including carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group
  • substituent groups including carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group
  • prefe ⁇ ed substitutent groups can be as described in the following paragraphs.
  • R t is selected from the following groups: hydrogen, oxygen, sulfur, amine, halide, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, substituted substitution group and combinations thereof.
  • Particularly prefe ⁇ ed R ⁇ is selected from the following groups: hydrogen, halide, thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, substituted substitution group and combinations thereof.
  • R ⁇ is especially preferably selected from the group consisting of alkyl, carbocyclic and substituted substitution group.
  • the substituted substitution group for R ⁇ are especially prefe ⁇ ed compounds or moieties such as:
  • Prefe ⁇ ed R 2 is selected from the following groups: hydrogen, oxygen, halide, carbonyl, alkyl, alkenyl, carbocyclic, substituted substitution group, and combinations thereof. Particularly prefe ⁇ ed R 2 is selected from the following groups: hydrogen, halide, alkyl, alkenyl, carbocyclic, substituted substitution group, and combinations thereof. R 2 is preferably selected from the group consisting of halide, alkyl and substituted substitution group.
  • the substituted substitution group for R 2 are especially prefe ⁇ ed compounds or moieties such as:
  • R 3 is selected from the following groups: hydrogen, oxygen, sulfur, amine, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group and combinations thereof.
  • R 3 is selected from the following groups: hydrogen, oxygen, amine, hydroxyl ( — OH), carbonyl, alkyl, acylamino, oximyl, hydrazyl, substituted substitution group and combinations thereof.
  • R 3 is preferably selected from the group consisting of carbonyl, acylamino, oximyl, hydrazyl, and substituted substitution group.
  • the substituted substitution group for R 3 are especially prefe ⁇ ed compounds or moieties such as:
  • R 4 and R 5 are independently selected from the following groups: hydrogen, oxygen, sulfur, phosphorus, amine, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, heterocyclic, acylamino, oximyl, hydrazyl, substituted substitution group and combinations thereof.
  • Particularly prefe ⁇ ed R ⁇ , and R 5 are independently selected from the following groups: hydrogen, oxygen, sulfur, amine, acidic, alkyl, substituted substitution group and combinations thereof.
  • R ⁇ , and R 5 are each preferably independently selected from the group consisting of oxygen, hydroxyl ( — OH), acidic, alkyl, and substituted substitution group.
  • the substituted substitution group for R ⁇ , and for R 5 are especially prefe ⁇ ed compounds or moieties such as:
  • Prefe ⁇ ed R 6 is selected from the following groups hydrogen, oxygen, amine, halide, hydroxyl ( — OH), acidic, alkyl, carbocyclic, acylamino, substituted substitution group and combinations thereof. Particularly prefe ⁇ ed R 6 is selected from the following groups: hydrogen, oxygen, amine, halide, hydroxyl ( — OH), acidic, alkyl, acylamino, substituted substitution group and combinations thereof. R 6 is preferably selected from the group consisting of amine, acidic, alkyl, and substituted substitution group.
  • the substituted substitution group for R 6 are especially prefe ⁇ ed compounds or moieties such as:
  • Prefe ⁇ ed R 7 is selected from the following groups: hydrogen, oxygen, sulfur, amine, halide, hydroxyl ( — OH), thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, heterocyclic, substituted substitution group and combinations thereof.
  • Particularly prefe ⁇ ed R 7 is selected from the following groups: hydrogen, halide, thiol ( — SH), carbonyl, acidic, alkyl, alkenyl, carbocyclic, substituted substitution group and combinations thereof.
  • R is preferably selected from the groups consisting of carbocyclic and substituted substitution group.
  • the substituted substitution group for R are especially prefe ⁇ ed compounds or moieties such as:
  • the inhibitor ofthe invention can comprise substituent groups wherein Ri through R are as follows: Ri is preferably selected from the group consisting of alkyl, carbocyclic and substituted substitution group; R 2 is preferably selected from the group consisting of halide, alkyl and substituted substitution group; R 3 is preferably selected from the group consisting of carbonyl, acylamino, oximyl, hydrazyl, and substituted substitution group; K and R 5 are each preferably independently selected from the group consisting of oxygen, hydroxyl ( — OH), acidic, alkyl, and substituted substitution group; R 6 is preferably selected from the group consisting of amine, acidic, alkyl, and substituted substitution group; and R is preferably selected from the groups consisting of carbocyclic and substituted substitution group.
  • indole glyoxamides are particularly useful as PL A inhibiting moieties in some embodiments.
  • PL A inhibiting moieties in some embodiments.
  • 2-(3-(2-amino-2-oxoacetyl)-l-(biphenyl- 2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] shown in Figure 2, alternatively refe ⁇ ed to herein as ILY-4001 and/or as methyl indoxam has been found to be an effective phospholipase inhibitor or inhibiting moiety.
  • This indole compound is represented by the structure below, as formula (V):
  • This compound has been shown, based on in-vitro assays, to have phospholipase activity for a number of PLA2 classes, and is a strong inhibitor of mouse and human PLA2IB enzymes in vitro (Singer, Ghomashchi et al. 2002; Smart, Pan et al. 2004).
  • This indole compound was synthesized (See, Example 4) and as noted above, was evaluated in-vivo for phospholipase- A2 inhibition in a mice model. (See, Example 5, including Examples 5A through 5C).
  • This indole compound was characterized with respect to inhibition activity, abso ⁇ tion and bioavailabihty. (See, Example 6, including Examples 6A through 6C).
  • indole compounds are also included within the scope of this invention. Many indoles have been described in the literature, for example, in connection with reported structure-activity-relationship studies (Schevitz, Bach et al. 1995; Dillard, Bach et al. 1996; Dillard, Bach et al. 1996; Draheim, Bach et al. 1996; Mihelich and Schevitz 1999). Table 1 lists various indole compounds, together with reported activity data against different phospholipase enzymes, including: human non- pancreatic PLA2 (hnp PLA2), human pancreatic secreted PLA2 (hps PLA2), and porcine pancreatic secreted PLA2 (pps PLA2). Table 1 : Indole Compounds
  • indole compounds can be employed within the scope of this invention. Table 2 lists some of such other indole compounds. Table 2: Indole Compounds Indole glyoxamides
  • indole-related compounds Other compounds having fused five-membered rings and six-membered rings with at least one heteroatom (refe ⁇ ed to herein generally as indole-related compounds) can also be used in connection with the present invention.
  • Table 3 lists some of such other indole-related compounds, and as relevant, patent references.
  • B and D are independently N or C
  • Z is Cyclohexenyl, phenyl, pyridyl, etc,.
  • indole-compounds ofthe invention can generally include "inverse indole compounds" that are minor-image analogues ofthe core structure ofthe co ⁇ esponding indole based on a reference axis taken orthogonal to and bisecting the fused bond between the five-membered and six- membered ring core, but that maintain the defined substituent groups at the same position. (See Figure 6C compared to Figure 6D).
  • Indole compounds and indole- related compounds ofthe invention can also include "reciprocal indole compounds" and "reciprocal indole-related compounds” that are minor-image analogues ofthe core structure ofthe co ⁇ esponding indole based on a reference axis taken along the axis of the fused bond between the five-membered and six-membered ring core, but which maintain at least each ofthe -R 3 and -R ⁇ , positions and each ofthe -R ⁇ and -R 7 at the same position, and that maintain -R and at least one of -R 5 and -R at the same position.
  • Representative pharmaceutically acceptable salts include but are not limited to, the alkali and alkaline earth salts such as lithium, sodium, potassium, calcium, magnesium, aluminum and the like. Salts are conveniently prepared from the free acid by treating the acid in solution with a base or by exposing the acid to an ion exchange resin. Included within the definition of pharmaceutically acceptable salts are the relatively non-toxic, inorganic and organic base addition salts of compounds ofthe present invention, for example, ammonium, quaternary ammonium, and amine cations, derived from nitrogenous bases of sufficient basicity to form salts with the compounds of this invention (see, for example, S. M. Berge, et al.,”Pharmaceutical Salts,"J. Phar.
  • the basic group (s) ofthe compound ofthe invention may be reacted with suitable organic or inorganic acids to form salts such as acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, chloride, edetate, edisylate, estolate, esylate, fluoride, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, bromide, chloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, malseate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate
  • suitable organic or inorganic acids to form salts such
  • phospholipase inhibitors ofthe present invention can modulate or inhibit (e.g., blunt or reduce) the catalytic activity of phospholipases, preferably phospholipases secreted or contained in the gastrointestinal tract, including the gastric compartment, and more particularly the duodenum and/or the small intestine.
  • such enzymes preferably include, but are not limited to, secreted Group IB phospholipase A 2 (PL A 2 -IB), also refe ⁇ ed to as pancreatic phospholipase A 2 (p-PL A 2 ) and herein refe ⁇ ed to as "PL A IB” or "phospholipase-A 2 IB.
  • Such enzymes can also include other phospholipase A2's secreted, such as Group IIA phospholipase A (PL A 2 IIA).
  • phospholipases can also be considered within the scope of invention, including for example: phospholipase Al (PLA ; phospholipase B (PLB); phospholipase C (PLC); and phospholipase D (PLD).
  • PKA phospholipase Al
  • PLB phospholipase B
  • PLC phospholipase C
  • PLD phospholipase D
  • the inhibitors ofthe invention preferably inhibit the activity at least the phospholipase- A 2 IB enzyme.
  • the inhibitors ofthe present invention are specific, or substantially specific for inhibiting phospholipase activity, such as phospholipase A activity (including for example phospholipase- A 2 IB).
  • phospholipase A activity including for example phospholipase- A 2 IB.
  • inhibitors ofthe present invention do not inhibit or do not significantly inhibit or essentially do not inhibit lipases, such as pancreatic triglyceride lipase (PTL) and carboxyl ester lipase (CEL).
  • PTL pancreatic triglyceride lipase
  • CEL carboxyl ester lipase
  • inhibitors ofthe present invention inhibit PL A 2 , and preferably phospholipase- A 2 IB, but in each case do not inhibit or do not significantly inhibit or essentially do not inhibit any other phospholipases; in some prefe ⁇ ed embodiments, inhibitors ofthe present invention inhibit PL A 2 , and preferably phospholipase- A IB, but in each case do not inhibit or do not significantly inhibit or essentially do not inhibit PLAi; in some prefe ⁇ ed embodiments, inhibitors ofthe present invention inhibit PL A 2 , and preferably phospholipase- A 2 IB, but do not inhibit or do not significantly inhibit or essentially do not inhibit PLB.
  • the phospholipase inhibitor does not act on the gastrointestinal mucosa, for example, it does not inhibit or does not significantly inhibit or essentially does not inhibit membrane-bound phospholipases.
  • the different activities of PL A 2 , PL Ai , and PLB are generally well- characterized and understood in the art.
  • PL A 2 hydrolyzes phospholipids at the sn-2 position liberating 1-acyl lysophospholipids and fatty acids; PL Ai acts on phospholipids at the sn-1 position to release 2-acyl lysophospholipids and fatty acids; and phospholipase B cleaves phospholipids at both sn-1 and sn-2 positions to form a glycerol and two fatty acids. See, e.g., Devlin, Editor, Textbook of Biochemistry with Clinical Co ⁇ elations, 5 th ed. Pp 1104-1110 (2002).
  • PLB are mostly ofthe phosphatidylcholine and phosphatidylethanolamine types, and can be of dietary or biliary origin, or may be derived from being sloughed off of cell membranes.
  • PL A acts at the sn-1 position to produce 2-acyl lysophosphatidylcholine and free fatty acid
  • PL A 2 acts at the sn-2 position to produce 1-acyl lysophosphatidylcholine and free fatty acid
  • PLB acts at both positions to produce glycerol 3-phosphorylcholine and two free fatty acids (Devlin, 2002).
  • Pancreatic PL A 2 (and phospholipase- A 2 IB) is secreted by acinar cells ofthe exocrine pancreas for release in the duodenum via pancreatic juice.
  • PL A 2 (and phospholipase- A IB) is secreted as a proenzyme, ca ⁇ ying a polypeptide chain that is subsequently cleaved by proteases to activate the enzyme's catalytic site.
  • Common features of PL A enzymes include sizes of about 13 to about 15 kDa; stability to heat; and 6 to 8 disulfides bridges. Common features of PL A enzymes also include conserved active site architecture and calcium-dependent activities, as well as a catalytic mechanism involving concerted binding of His and Asp residues to water molecules and a calcium cation, in a His-calcium-Asp triad.
  • a phospholipid substrate can access the catalytic site by its polar head group through a slot enveloped by hydrophobic and cationic residues (including lysine and arginine residues) described in more detail below.
  • the multi-coordinated calcium ion activates the acyl carbonyl group ofthe sn-2 position ofthe phospholipid substrate to bring about hydrolysis (Devlin, 2002).
  • inhibitors ofthe present invention inhibit this catalytic activity of PL A 2 by interacting with its catalytic site.
  • PL A 2 enzymes are active for catabolizing phospholipids substrates primarily at the lipid-water interface of lipid aggregates found in the gastrointestinal lumen, including, for example, fat globules, emulsion droplets, vesicles, mixed micelles, and/or disks, any one of which may contain triglycerides, fatty acids, bile acids, phospholipids, phosphatidylcholine, lysophospholipids, lysophosphatidylcholine, cholesterol, cholesterol esters, other amphiphiles and/or other diet metabolites. Such enzymes can be considered to act while "docked" to a lipid-water interface.
  • the phospholipid substrates are typically a ⁇ anged in a mono layer or in a bilayer, together with one or more other components listed above, which form part of the outer surface ofthe aggregate.
  • the surface of a phospholipase bearing the catalytic site contacts this interface facilitating access to phospholipid substrates.
  • This surface of the phospholipase is known as the -face, i.e., the interfacial recognition face ofthe enzyme.
  • the structural features ofthe t-face of PL A 2 have been well documented. See, e.g., Jain, M.K, et al, Methods in Enzymology, vol.239, 1995, 568-614, incorporated herein by reference.
  • the inhibitors ofthe present invention can take advantage of these structural features to inhibit PL A 2 activity.
  • the aperture ofthe slot forming the catalytic site is normal to the t-face plane.
  • the aperture is su ⁇ ounded by a first crown of hydrophobic residues (mainly leucine and isoleucine residues), which itself is contained in a ring of cationic residues (including lysine and arginine residues).
  • PL A 2 enzymes share a conserved active site architecture and a catalytic mechanism involving concerted binding of His and Asp residues to water molecules and a calcium cation.
  • a phospholipid substrate can access the catalytic site of such enzymes with its polar head group directed through a slot enveloped by hydrophobic and cationic residues.
  • the multi-coordinated calcium ion activates the acyl carbonyl group ofthe sn-2 position ofthe phospholipid substrate to bring about hydrolysis.
  • Example 5 In view ofthe substantial structure-activity-relationship studies for phospholipase- A2 enzymes, considered together with the significant experimental data demonstrated in Example 5 (including Examples 5A through 5C), a skilled person can appreciate that the observed inhibitive effect of ILY-4001 can be realized in other indole compounds ofthe invention (e.g., having the identical core structure) as well as in indole-related compounds comprising a fused five-membered ring and six-membered ring.
  • substituents at positions 3 and 4 and 5 ofthe indole structure can be selected and evaluated to be effective for polar interaction with the enzyme and with calcium ion (associated with the calcium-dependent phospholipase activity).
  • substituents at positions 1 and 2 ofthe indole structure can be selected and evaluated to be relatively hydrophobic.
  • the polar groups at positions 3, 4 and 5 and the relatively hydrophobic groups at positions 1 and 2 can effectively associate the inhibitor (or inhibiting moiety) with a hydrophilic lipid-water interface (via the hydrophobic regions), and also orient the inhibitor (or inhibiting moiety) such that its polar region can be effectively positioned into the enzyme pocket - with its polar head group directed through a slot enveloped by hydrophobic and cationic residues.
  • co ⁇ esponding groups on the indole-related compound shown therein can have the same functionality.
  • R 5 ofthe indole- related structure can be selected and evaluated to be effective for polar interaction with the enzyme and with calcium ion, and that the substituents at positions R 1 and R 2 ofthe indole-related structure can be selected and evaluated to be relatively hydrophobic.
  • the above-described inverse indole compounds that are mi ⁇ or-image analogues ofthe core structure ofthe co ⁇ esponding indole of interest, and the above-described reciprocal indole compounds and reciprocal indole-related compounds that are alternative mi ⁇ or-image analogues of the core structure ofthe co ⁇ esponding indole or related compound can be similarly configured with polar substituents and hydrophobic substituents to provide alternative indole structures and alternative indole-related structures within the scope ofthe invention.
  • a person skilled in the art can evaluate particular inhibitors within the scope of this invention using known assaying and evaluation approaches.
  • the extent of inhibition ofthe inhibitors ofthe invention can be evaluated using in-vitro assays (See, for example, Example 6A) and/or in-vivo studies (See, for example, Example 5). Further, binding of a phospholipase inhibitor to a phospholipase enzyme can be evaluated by nuclear magnetic resonance, for example to provide identification of sites essential or non-essential for such binding interaction. Additionally, one of skill in the art can use available structure-activity relationship (SAR) for phospholipase inhibitors that suggest positions where structural variations are allowed.
  • SAR structure-activity relationship
  • a library of candidate phospholipase inhibitors can be designed to feature different points of attachment ofthe phospholipase inhibiting moiety, e.g., chosen based on information described above as well as randomly, so as to present the phospholipase inhibiting moiety in multiple distinct orientations.
  • Candidates can be evaluated for phospholipase inhibiting activity to obtain phospholipase inhibitors with suitable attachment points ofthe phospholipase inhibiting moiety to the polymer moiety or other non-absorbed moiety.
  • the extent of inhibition is not na ⁇ owly critical to the invention, but can be of significance in particular embodiments.
  • the term "inhibits" and its grammatical variations are not intended to require a complete inhibition of enzymatic activity.
  • it can refer to a reduction in enzymatic activity by at least about 50%, at least about 75%, preferably by at least about 90%, more preferably at least about 98%, and even more preferably at least about 99% ofthe activity ofthe enzyme in the absence ofthe inhibitor.
  • the phrase "does not inhibit” and its grammatical variations does not require a complete lack of effect on the enzymatic activity. For example, it refers to situations where there is less than about 20%, less than about 10%, less than about 5%, preferably less than about 2%, and more preferably less than about 1% of reduction in enzyme activity in the presence ofthe inhibitor. Most preferably, it refers to a minimal reduction in enzyme activity such that a noticeable effect is not observed.
  • the phrase “does not significantly inhibit” and its grammatical variations refers to situations where there is less than about 40%, less than about 30%, less than about 25%, preferably less than about 20%, and more preferably less than about 15% of reduction in enzyme activity in the presence ofthe inhibitor. Further, the phrase “essentially does not inhibit” and its grammatical variations refers to situations where there is less than about 30%, less than about 25%, less than about 20%, preferably less than about 15 %, and more preferably less than about 10% of reduction in enzyme activity in the presence ofthe inhibitor.
  • the inhibitors can modulate phospholipase activity by reversible and/or i ⁇ eversible inhibition.
  • Reversible inhibition by a phospholipase inhibitor ofthe present invention may be competitive (e.g. where the inhibitor binds to the catalytic site of a phospholipase), noncompetitive (e.g., where the inhibitor binds to an allosteric site of a phospholipase to effect an allosteric change), and/or uncompetitive (where the inhibitor binds to a complex between a phospholipase and its substrate).
  • Inhibition may also be i ⁇ eversible, where the phospholipase inhibitor remains bound, or significantly remains bound, or essentially remains bound to a site on a phospholipase without dissociating, without significantly dissociating, or essentially without dissociating from the enzyme.
  • Methods of Treating Phospholipase-Related Conditions [0074] The present invention provides methods of treating phospholipase- related conditions. In prefe ⁇ ed embodiments, the inhibitor can be localized in a gastrointestinal lumen.
  • phospholipase-related condition refers to a condition in which modulating the activity and/or re-absorption of a phospholipase, and/or modulating the production and/or effects of one or more products ofthe phospholipase, is desirable.
  • an inhibitor ofthe present invention reduces the activity and/or re-absorption of a phospholipase, and/or reduces the production and/or effects of one or more products ofthe phospholipase.
  • phospholipase A2-related condition refers to a condition in which modulating the activity and/or re-absorption of phospholipase A2 is desirable and/or modulating the production and/or effects of one or more products of phospholipase A2 activity is desirable.
  • an inhibitor ofthe present invention reduces the activity and/or re-absorption of phospholipase A2, and/or reduces the production and/or effects of one or more products ofthe phospholipase A2.
  • phospholipase A2-related conditions include, but are not limited to, insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity) and/or cholesterol- related conditions, and any combination thereof.
  • the present invention provides methods, pharmaceutical compositions, and kits for the treatment of animal subjects.
  • the term "animal subject” as used herein includes humans as well as other mammals.
  • the mammals can be selected from mice, rats, rabbits, guinea pigs, hamsters, cats, dogs, porcine, poultry, bovine and horses, as well as combinations thereof.
  • the term "treating" as used herein includes achieving a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration ofthe underlying disorder being treated.
  • therapeutic benefit includes eradication or amelioration ofthe underlying diabetes.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more ofthe physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding the fact that the patient may still be afflicted with the underlying disorder.
  • reducing PL A activity can provide therapeutic benefit not only when insulin resistance is co ⁇ ected, but also when an improvement is observed in the patient with respect to other disorders that accompany diabetes like fatigue, blurred vision, or tingling sensations in the hands or feet.
  • a phospholipase inhibitor ofthe present invention may be administered to a patient at risk of developing a phospholipase-related condition, e.g., diabetes, obesity, or hypercholesterolemia, or to a patient reporting one or more ofthe physiological symptoms of such conditions, even though a diagnosis may not have been made.
  • a phospholipase-related condition e.g., diabetes, obesity, or hypercholesterolemia
  • the present invention provides compositions comprising a phospholipase inhibitor.
  • the inhibitor is not absorbed through a gastrointestinal mucosa and/or that is localized in a gastrointestinal lumen as a result of efflux from a gastrointestinal mucosal cell.
  • the phospholipase inhibitors of the present invention produce a benefit, including either a prophylactic benefit, a therapeutic benefit, or both, in treating one or more conditions by inhibiting phospholipase activity.
  • the methods for effectively inhibiting phospholipase described herein can apply to any phospholipase-related condition, that is, to any condition in which modulating the activity and/or re-absorption of a phospholipase, and/or modulating the production and/or effects of one or more products ofthe phospholipase, is desirable.
  • such conditions include phospholipase- A 2 -related conditions and/or phospholipase A2-related conditions induced by diet, that is, conditions which are brought on, accelerated, exacerbated, or otherwise influenced by diet.
  • Phospholipase- A 2 -related conditions include, but are not limited to, diabetes, weight gain, and cholesterol-related conditions, as well as hyperlipidemia, hypercholesterolemia, cardiovascular disease (such as heart disease and stroke), hypertension, cancer, sleep apnea, osteoarthritis, gallbladder disease, fatty liver disease, diabetes type 2 and other insulin-related conditions.
  • one or more of these conditions may be produced as a result of consumption of a high fat or Western diet; in some embodiments, one or more of these conditions may be produced as a result of genetic causes, metabolic disorders, environmental factors, behavioral factors, or any combination of these.
  • some embodiments ofthe invention relate to one or more of a high-carbohydrate diet, a high-saccharide diet, a high-fat diet and/or a high-cholesterol diet, in various combinations.
  • Such diets are generally refe ⁇ ed to herein as a "high-risk diets" (and can include ,for example, Western diets).
  • Such diets can heighten the risk profile of a subject patient for one or more conditions, including an obesity-related condition, an insulin-related condition and/or a cholesterol-related condition.
  • such high-risk diets can, in some embodiments, include at least a high- carbohydrate diet together with one or more of a high-saccharide diet, a high-fat diet and/or a high-cholesterol diet.
  • a high-risk diet can also include a high-saccharide diet in combination with one or both of a high-fat diet and/or a high-cholesterol diet.
  • a high-risk diet can also comprise a high-fat diet in combination with a high-cholesterol diet.
  • a high-risk diet can include the combination of a high- carbohydrate diet, a high-saccharide diet and a high-fat diet.
  • a high-risk diet can include a high-carbohydrate diet, a high-saccharide diet, and a high- cholesterol diet.
  • a high-risk diet can include a high-carbohydrate diet, a high-fat diet and a high-cholesterol diet.
  • a high-risk diet can include a high-saccharide diet, a high-fat diet and a high-cholesterol diet.
  • a high-risk diet can include a high-carbohydrate diet, a high- saccharide diet, a high- fat diet and a high-cholesterol diet.
  • the diet of a subject can comprise a total caloric content, for example, a total daily caloric content.
  • the subject diet can be a high-fat diet.
  • at least about 50% ofthe total caloric content can come from fat.
  • at least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from fat.
  • at least about 15% or at least about 10% ofthe total caloric content can come from fat.
  • the diet can be a high-carbohydrate diet.
  • at least about 50% ofthe total caloric content can come from carbohydrates.
  • at least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from carbohydrates.
  • at least about 15% or at least about 10% ofthe total caloric content can come from carbohydrate.
  • the diet can be a high-saccharide diet.
  • at least about 50% ofthe total caloric content can come from saccharides.
  • at least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from saccharides.
  • at least about 15% or at least about 10% ofthe total caloric content can come from saccharides.
  • the diet can be a high-cholesterol diet, h such embodiments, the diet can comprise at least about 1 % cholesterol (wt/wt, relative to fat). In other such embodiments, the diet can comprise at least about 0.5 % or at least about 0.3 % or at least about 0.1 %, or at least about 0.07 % cholesterol (wt/wt relative to fat). In some embodiments, in which a high-cholesterol diet is combined with one or more of a high-fat diet, a high-carbohydrate diet or a high-saccharide diet, the diet can comprise at least about 0.05 % or at least about 0.03 % cholesterol (wt/wt, relative to fat).
  • a high fat diet can include, for example, diets high in meat, dairy products, and alcohol, as well as possibly including processed food stuffs, red meats, soda, sweets, refined grains, deserts, and high-fat dairy products, for example, where at least about 25% of calories come from fat and at least about 8% come from saturated fat; or at least about 30% of calories come from fat and at least about 10% come from saturated fat; or where at least about 34% of calories came from fat and at least about 12% come from saturated fat; or where at least about 42% of calories come from fat and at least about 15% come from saturated fat; or where at least about 50% of calories come from fat and at least about 20% come from saturated fat.
  • One such high fat diet is a "Western diet" which refers to the diet of industrialized countries, including, for example, a typical American diet, Western European diet, Australian diet, and/or Japanese diet.
  • a Western diet comprises at least about 17% fat and at least about 0.1% cholesterol (wt/wt); at least about 21% fat and at least about 0.15% cholesterol (wt/wt); or at least about 25% and at least about 0.2% cholesterol (wt/wt).
  • Such high-risk diets may include one or more high-risk foodstuffs.
  • some embodiments ofthe invention relate to one or more of a high-carbohydrate foodstuff, a high- saccharide foodstuff, a high-fat foodstuff and/or a high-cholesterol foodstuff, in various combinations.
  • Such foodstuffs are generally refe ⁇ ed to herein as a "high-risk foodstuffs" (including for example Western foodstuffs).
  • Such foodstuffs can heighten the risk profile of a subject patient for one or more conditions, including an obesity- related condition, an insulin-related condition and/or a cholesterol-related condition.
  • such high-risk foodstuffs can, in some embodiments, include at least a high- carbohydrate foodstuff together with one or more of a high-saccharide foodstuff, a high-fat foodstuff and or a high-cholesterol foodstuff.
  • a high-risk foodstuff can also include a high-saccharide foodstuff in combination with one or both of a high-fat foodstuff and/or a high-cholesterol foodstuff.
  • a high-risk foodstuff can also comprise a high-fat foodstuff in combination with a high-cholesterol foodstuff.
  • a high-risk foodstuff can include the combination of a high-carbohydrate foodstuff, a high-saccharide foodstuff and a high-fat foodstuff.
  • a high-risk foodstuff can include a high-carbohydrate foodstuff, a high-saccharide foodstuff, and a high-cholesterol foodstuff, hi other embodiments, a high-risk foodstuff can include a high-carbohydrate foodstuff, a high- fat foodstuff and a high-cholesterol foodstuff.
  • a high-risk foodstuff can include a high- saccharide foodstuff, a high-fat foodstuff and a high-cholesterol foodstuff.
  • a high-risk foodstuff can include a high-carbohydrate foodstuff, a high- saccharide foodstuff, a high- fat foodstuff and a high-cholesterol foodstuff.
  • the food product composition can comprise a foodstuff having a total caloric content.
  • the food-stuff can be a high-fat foodstuff. In such embodiments, at least about 50% ofthe total caloric content can come from fat.
  • At least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from fat.
  • a high-fat foodstuff in which a high-fat foodstuff is combined with one or more of a high- carbohydrate foodstuff, a high-saccharide foodstuff or a high-cholesterol foodstuff, at least about 15% or at least about 10% ofthe total caloric content can come from fat.
  • the food-stuff can be a high- carbohydrate foodstuff.
  • at least about 50% ofthe total caloric content can come from carbohydrates.
  • At least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from carbohydrates.
  • a high- carbohydrate foodstuff in which a high- carbohydrate foodstuff is combined with one or more of a high-fat foodstuff, a high- saccharide foodstuff or a high-cholesterol foodstuff, at least about 15% or at least about 10% ofthe total caloric content can come from carbohydrate.
  • the food-stuff can be a high-saccharide foodstuff.
  • at least about 50% ofthe total caloric content can come from saccharides.
  • At least about 40%, or at least about 30% or at least about 25%, or at least about 20% ofthe total caloric content can come from saccharides.
  • at least about 15% or at least about 10% ofthe total caloric content can come from saccharides.
  • the food-stuff can be a high-cholesterol foodstuff.
  • the food-stuff can comprise at least about 1 % cholesterol (wt/wt, relative to fat).
  • the foodstuff can comprise at least about 0.5 %, or at least about 0.3 % or at least about 0.1 %, or at least about 0.07 % cholesterol (wt/wt relative to fat).
  • the foodstuff in which a high- cholesterol foodstuff is combined with one or more of a high-fat foodstuff, a high- carbohydrate foodstuff or a high-saccharide foodstuff, can comprise at least about 0.05 % or at least about 0.03 % cholesterol (wt/wt, relative to fat).
  • the methods ofthe invention can be used advantageously together with other methods, including for example methods broadly directed to treating insulin-related conditions, weight-related conditions and/or cholesterol-related conditions (including dislipidemia generally) and any combination thereof. Aspects of such conditions are described below. Treatment of Insulin-Related Conditions
  • insulin-related disorders refers to a condition such as diabetes where the body does not produce and/or does not properly use insulin.
  • a patient is diagnosed with pre-diabetes or diabetes by using a Fasting Plasma Glucose Test (FPG) and or an Oral Glucose Tolerance Test (OGTT).
  • FPG Fasting Plasma Glucose Test
  • OGTT Oral Glucose Tolerance Test
  • a fasting blood glucose level between about 100 and about 125 mg/dl can indicate pre-diabetes; while a person with a fasting blood glucose level of about 126 mg/dl or higher can indicate diabetes.
  • a patient's blood glucose level can be measured after a fast and two hours after drinking a glucose-rich beverage.
  • a two-hour blood glucose level between about 140 and about 199 mg/dl can indicate pre-diabetes; while a two-hour blood glucose level at about 200 mg/dl or higher can indicate diabetes.
  • a lumen localized phospholipase inhibitor ofthe present invention produces a benefit in treating an insulin-related condition, for example, diabetes, preferably diabetes type 2.
  • benefits may include, but are not limited to, increasing insulin sensitivity and improving glucose tolerance.
  • Other benefits may include decreasing fasting blood insulin levels, increasing tissue glucose levels and/or increasing insulin-stimulated glucose metabolism.
  • these benefits may result from a number of effects brought about by reduced PL A 2 activity, including, for example, reduced membrane transport of phospholipids across the gastrointestinal mucosa and/or reduced production of 1-acyl lysophospholipids, such as 1-acyl lysophosphatydylcholine and/or reduced transport of lysophospholipids, 1-acyl lysophosphatydylcholine, that may act as a signaling molecule in subsequent pathways involved in diabetes or other insulin-related conditions.
  • 1-acyl lysophospholipids such as 1-acyl lysophosphatydylcholine and/or reduced transport of lysophospholipids, 1-acyl lysophosphatydylcholine
  • a lumen-localized phospholipase inhibitor is used that inhibits phospholipase A2 but does not inhibit or does not significantly inhibit or essentially does not inhibit phospholipase B.
  • the phospholipase inhibitor inhibits phospholipase A2 but no other gastrointestinal phospholipase, including not inhibiting or not significantly inhibiting or essentially not inhibiting phospholipase Al, and not inhibiting or not significantly inhibiting or essentially not inhibiting phospholipase.
  • weight-related conditions refers to unwanted weight gain, including overweight, obese and/or hyperlipidemic conditions, and in particular weight gain caused by a high fat or Western diet.
  • body mass index BMI
  • An adult is considered overweight if, for example, he or she has a body mass index of at least about 25, and is considered obese with a BMI of at least about 30.
  • a lumen localized phospholipase A2 inhibitor of the present invention can be used to treat weight-related conditions, including unwanted weight gain and/or obesity.
  • a lumen localized phospholipase A2 inhibitor decreases fat absorption after a meal typical of a Western diet.
  • a lumen localized phospholipase A2 inhibitor increases lipid excretion from a subject on a Western diet.
  • the phospholipase inhibitor reduces weight gain in a subject on a (typical) Western diet
  • practice ofthe present invention can preferentially reduce weight gain in certain tissues and organs, e.g., in some embodiments, a phospholipase A2 inhibitor can decrease weight gain in white fat of a subject on a Western diet.
  • these benefits may result from a number of effects brought about by reduced PL A 2 activity.
  • inhibition of PL A activity may reduce transport of phospholipids through the gastrointestinal lumen, for example, through the small intestine apical membrane, causing a depletion ofthe pool of phospholipids (e.g. phosphatidylcholine) in enterocytes, particularly in mammals fed with a high fat diet.
  • the de novo synthesis of phospholipids may not be sufficient to sustain the high turnover of phospholipids, e.g.
  • phosphatidylcholine needed to carry triglycerides, for example by transport in chylomicrons (See Tso, in Fat Absorption, 1986, chapt.6177-195, Kuksis A., Ed.), incorporated herein by reference.
  • PL A 2 inhibition can also reduce production of 1-acyl lysophospholipids, such as 1-acyl lysophosphatydylcholine, that may act as a signaling molecule in subsequent up-regulation pathways of fat absorption, including, for example the release of additional digestive enzymes or hormones, e.g., secretin.
  • 1-acyl lysophospholipids such as 1-acyl lysophosphatydylcholine
  • additional digestive enzymes or hormones e.g., secretin.
  • compositions, kits and methods for reducing or delaying the onset of diet-induced diabetes through weight gain can produce not only weight gain, but also can contribute to diabetic insulin resistance. This resistance may be recognized by decreased insulin and leptin levels in a subject.
  • the phospholipase inhibitors, compositions, kits and methods disclosed herein can be used in the prophylactic treatment of diet-induced diabetes, or other insulin-related conditions, e.g. in decreasing insulin and/or leptin levels in a subject on a Western diet.
  • a lumen-localized phospholipase inhibitor is used that inhibits phospholipase A2 but does not inhibitor or does not significantly inhibit or essentially does not inhibit phospholipase B.
  • the phospholipase inhibitor inhibits phospholipase A2 but no other gastrointestinal phospholipase, including not inhibiting or not significantly inhibiting or essentially not inhibiting phospholipase Al, and not inhibiting or not significantly inhibiting or essentially not inhibiting phospholipase B.
  • cholesterol-related conditions refers generally to a condition in which modulating the activity of HMG-CoA reductase is desirable and/or modulating the production and/or effects of one or more products of HMG-CoA reductase is desirable, and can in any case, include dislipidemia generally.
  • a phospholipase inhibitor ofthe present invention reduces the activity of HMG-CoA reductase and/or reduces the production and/or effects of one or more products of HMG-CoA reductase.
  • a cholesterol-related condition may involve elevated levels of cholesterol, in particular, non-HDL cholesterol in plasma (e.g., elevated levels of LDL cholesterol and/or VLDL/LDL levels).
  • non-HDL cholesterol in plasma e.g., elevated levels of LDL cholesterol and/or VLDL/LDL levels.
  • a patient is considered to have high or elevated cholesterol levels based on a number of criteria, for example, see Pearlman BL, The New Cholesterol Guidelines, Postgrad Med, 2002; 112(2):13-26, incorporated herein by reference. Guidelines include serum lipid profiles, such as LDL compared with HDL levels.
  • Examples of cholesterol-related conditions include hypercholesterolemia, lipid disorders such as hyperlipidemia, and atherogenesis and its sequelae of cardiovascular diseases, including atherosclerosis, other vascular inflammatory conditions, myocardial infarction, ischemic stroke, occlusive stroke, and peripheral vascular diseases, as well as other conditions in which decreasing cholesterol can produce a benefit.
  • Other cholesterol-related conditions of particular interest include dislipidemia conditions, such as hypertriglyceridemia. Hepatic triglyceride synthesis is regulated by available fatty acids, glycogen stores, and the insulin versus glucagon ratio.
  • Patients with a high glucose diet are likely to have a balance of hormones that maintains an excess of insulin and also build up glycogen stores, both of which enhance hepatic triglyceride synthesis.
  • diabetic patients are particularly susceptible, since they are often overweight and are in a state of caloric excess.
  • the present invention is particularly of interest, in each embodiment herein described, with respect to treatments directed to hypertriglyceridemia.
  • the phospholipase A2 inhibitors ofthe present invention can modulate tryglycerides and cholesterol through more than one mechanistic path.
  • the phospholipase A2 inhibitors ofthe invention can modulate cholesterol absorption and triglyceride absorption from the gastrointestinal tract, and can also modulate the metabolism of fat and glucose, for example, via signaling molecules such as lysophosphatidylcholine (the reaction product of PLA2 catalyzed hydrolysis of phosphatidylcholine), operating directly and/or in conjunction with other hormones such as insulin.
  • VLDL is a lipoprotein packaged by the liver for endogenous circulation from the liver to the peripheral tissues.
  • VLDL contains triglycerides, cholesterol, and phospholipase at its core along with apolipoproteins B100, CI, CII, CIII, and E at its perimeter.
  • Triglycerides make up more than half of VLDL by weight and the size of VLDL is determined by the amount of triglyceride.
  • VLDL Very large VLDL is secreted by the liver in states of caloric excess, in diabetes mellitus, and after alcohol consumption, because excess triglycerides are present.
  • inhibition of phospholipase A2 activity can impact metabolism, including for example hepatic triglyceride synthesis.
  • Modulated (e.g., reduced or at least relatively reduced increase) in triglyceride synthesis can provide a basis for modulating serum triglyceride levels and/or serum cholesterol levels, and further can provide a basis for treating hypertriglyceridemia and/or hypercholesterolemia.
  • Such treatments would be beneficial to both diabetic patients (who typically replace their carbohydrate restrictions with higher fat meals), and to hypertriglyceridemic patients (who typically substitute fat with high carbohydrate meals).
  • increased protein meals alone are usually not sustainable in the long term for most diabetic and/or hypertriglyceridemic patients.
  • the modulation of serum triglyceride levels can have a beneficial effect on cardiovascular diseases such as atherosclerosis.
  • VLDL remnants can either enter the liver (the large ones preferentially do this) or can give rise to LDL. Hence, elevated VLDL in the circulation lowers HDL, which is responsible for reverse cholesterol transport. Since hypertriglyceridemia contributes to elevated LDL levels and also contributes to lowered HDL levels, hypertriglyceridemia is a risk factor for cardiovascular diseases such as atherosclerosis and coronary artery disease (among others, as noted above). Accordingly, modulating hypertriglyceridemia using the phospholipase- A2 inhibitors ofthe present invention also provide a basis for treating such cardiovascular diseases.
  • compositions, kits, and methods ofthe present invention include those cu ⁇ ently treated with statins, as well as other conditions in which decreasing cholesterol absorption can produce a benefit.
  • a lumen-localized phospholipase inhibitor ofthe present invention can be used to reduce cholesterol levels, in particular non-HDL plasma cholesterol levels, as well as to treat hypertriglyceridemia.
  • the composition can inhibit phospholipase A2 and at least one other gastrointestinal phospholipase in addition to phospholipase A2, such as preferably phospholipase B, and also such as phospholipase Al, phospholipase C, and/or phospholipase D.
  • the differential activities of phospholipases can be used to treat certain phospholipase-related conditions without undesired side effects resulting from inhibiting other phospholipases.
  • a phospholipase inhibitor that inhibits PL A but not inhibiting or not significantly inhibiting or essentially not inhibiting, for example, PLAl, PLB, PLC, or PLD can be used to treat an insulin-related condition (e.g. diabetes) and/or a weight- related condition (e.g.
  • the phospholipase inhibitors, methods, and kits disclosed herein can be used in the treatment of phospholipase-related conditions. In some prefe ⁇ ed embodiments, these effects can be realized without a change in diet and/or activity on the part ofthe subject. For example, the activity of PL A 2 in the gastrointestinal lumen may be inhibited to result in a decrease in fat absorption and/or a reduction in weight gain in a subject on a Western diet compared to if the subject was not receiving PL A 2 inhibiting treatment.
  • this decrease and/or reduction occurs without a change, without a significant change, or essentially without a change, in energy expenditure and/or food intake on the part ofthe subject, and without a change, or without a significant change, or essentially without a change in the body temperature of the subject.
  • a phospholipase inhibitor ofthe present invention can be used to offset certain negative consequences of high fat diets without affecting normal aspects of metabolism on non-high fat diets.
  • the present invention also includes kits that can be used to treat phospholipase-related conditions, preferably phospholipase A2-related conditions or phospholipase-related conditions induced by diet, including, but not limited to, insulin- related conditions (e.g., diabetes, particularly diabetes type 2), weight-related conditions (e.g., obesity) and/or cholesterol-related conditions.
  • kits that can be used to treat phospholipase-related conditions, preferably phospholipase A2-related conditions or phospholipase-related conditions induced by diet, including, but not limited to, insulin- related conditions (e.g., diabetes, particularly diabetes type 2), weight-related conditions (e.g., obesity) and/or cholesterol-related conditions.
  • kits comprise at least one composition ofthe present invention and instructions teaching the use ofthe kit according to the various methods described herein.
  • the phospholipase inhibitors useful in the present invention, or pharmaceutically acceptable salts thereof, can be delivered to a patient using a number of routes or modes of administration.
  • pharmaceutically acceptable salt means those salts which retain the biological effectiveness and properties ofthe compounds used in the present invention, and which are not biologically or otherwise undesirable.
  • Such salts include salts with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid.
  • suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine and triethanolamine.
  • the phospholipase inhibitor may be administered in combination with one or more other therapeutic agents.
  • the choice of therapeutic agent that can be co-administered with a composition ofthe invention will depend, in part, on the condition being treated.
  • a phospholipase inhibitor of some embodiments ofthe present invention can be used in combination with a statin, a fibrate, a bile acid binder, an ezitimibe (e.g., Zetia, etc), a saponin, a lipase inhibitor (e.g. Orlistat, etc), and/or an appetite suppressant, and the like.
  • a phospholipase inhibitor of some embodiments the present invention can be used in combination with a biguanide (e.g., Metformin), thiazolidinedione, and/or - glucosidase inhibitor, and the like.
  • a biguanide e.g., Metformin
  • thiazolidinedione e.g., thiazolidinedione
  • - glucosidase inhibitor e.g., glucosidase inhibitor
  • the phospholipase inhibitors may be administered per se or in the form of a pharmaceutical composition wherein the active compound(s) is in admixture or mixture with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers compromising excipients and auxiliaries which facilitate processing ofthe active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the phospholipase inhibitors can be administered by direct placement, orally, and/or rectally.
  • the phospholipase inhibitor or the pharmaceutical composition comprising the phospholipase inhibitor is administered orally.
  • the oral form in which the phospholipase inhibitor is administered can include a powder, tablet, capsule, solution, or emulsion.
  • the effective amount can be administered in a single dose or in a series of doses separated by appropriate time intervals, such as hours.
  • the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, wafers, and the like, for oral ingestion by a patient to be treated.
  • the inhibitor may be formulated as a sustained release preparation.
  • Pharmaceutical preparations for oral use can be obtained as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, mehtyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl py ⁇ olidone (PVP).
  • disintegrating agents maybe added, such as the cross-linked polyvinyl py ⁇ olidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores can be provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl py ⁇ olidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the oral formulation does not have an enteric coating.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers, hi soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for administration.
  • Suitable carriers used in formulating liquid dosage forms for oral as well as parenteral administration include non-aqueous, pharmaceutically-acceptable polar solvents such as hydrocarbons, alcohols, amides, oils, esters, ethers, ketones, and/or mixtures thereof, as well as water, saline solutions, electrolyte solutions, dextrose solutions (e.g., DW5), and/or any other aqueous, pharmaceutically acceptable liquid.
  • non-aqueous, pharmaceutically-acceptable polar solvents such as hydrocarbons, alcohols, amides, oils, esters, ethers, ketones, and/or mixtures thereof, as well as water, saline solutions, electrolyte solutions, dextrose solutions (e.g., DW5), and/or any other aqueous, pharmaceutically acceptable liquid.
  • Suitable nonaqueous, pharmaceutically-acceptable polar solvents include, but are not limited to, alcohols (e.g., aliphatic or aromatic alcohols having 2-30 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, t-butanol, hexanol, octanol, benzyl alcohol, amylene hydrate, glycerin (glycerol), glycol, hexylene glycol, lauryl alcohol, cetyl alcohol, stearyl alcohol, tetrahydrofurfuryl alcohol, fatty acid esters of fatty alcohols such as polyalkylene glycols (e.g., polyethylene glycol and/or polypropylene glycol), sorbitan, cholesterol, sucrose and the like); amides (e.g., dimethylacetamide (DMA), benzyl benzoate DMA, N,N-dimethylacetamide amides
  • Formulations for rectal administration may be prepared in the form of a suppository, an ointment, an enema, a tablet, or a cream for release ofthe phospholipase inhibitor in the gastrointestinal tract, e.g., the small intestine.
  • Rectal suppositories can be made by mixing one or more phospholipase inhibitors ofthe present invention, or pharmaceutically acceptable salts thereof, with acceptable vehicles, for example, cocoa butter, with or without the addition of waxes to alter melting point.
  • Acceptable vehicles can also include glycerin, salicylate and/or polyethylene glycol, which is solid at normal storage temperature, and a liquid at those temperatures suitable to release the phospholipase inhibitor inside the body, such as in the rectum. Oils may also be used in rectal formulations ofthe soft gelatin type and in suppositories.
  • Water soluble suppository bases such as polyethylene glycols of various molecular weights, may also be used.
  • Suspension formulations may be prepared that use water, saline, aqueous dextrose and related sugar solutions, and glycerols, as well as suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.
  • Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are present in an effective amount, i.e., in an amount sufficient to produce a therapeutic and/or a prophylactic benefit in at least one condition being treated. The actual amount effective for a particular application will depend on the condition being treated and the route of administration.
  • an effective amount is well within the capabilities of those skilled in the art, especially in light ofthe disclosure herein.
  • the IC50 values and ranges provided in Table 1 above provide guidance to enable one of ordinary skill in the art to select effective dosages ofthe co ⁇ esponding phospholipase inhibiting moieties.
  • the effective amount when referring to a phospholipase inhibitor will generally mean the dose ranges, modes of administration, formulations, etc., that have been recommended or approved by any ofthe various regulatory or advisory organizations in the medical or pharmaceutical arts (eg, FDA, AMA) or by the manufacturer or supplier. Effective amounts of phospholipase inhibitors can be found, for example, in the Physicians Desk Reference.
  • the effective amount when referring to producing a benefit in treating a phospholipase-related condition such as insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity), and/or cholesterol related-conditions will generally mean the levels that achieve clinical results recommended or approved by any ofthe various regulatory or advisory organizations in the medical or pharmaceutical arts (eg, FDA, AMA) or by the manufacturer or supplier.
  • a person of ordinary skill using techniques known in the art can determine the effective amount ofthe phospholipase inhibitor.
  • the effective amount of a phospholipase inhibitor localized in the gastsrointestinal lumen can be less than the amount administered in the absence of such localization.
  • the phospholipase inhibitor reduces activity of phospholipase to a greater extent compared to non-lumen localized inhibitors. Lumen-localization ofthe phospholipase inhibitor can decrease the effective amount necessary for the treatment of phospholipase-related conditions, such as insulin-related conditions (e.g., diabetes), weight-related conditions (e.g., obesity) and/or cholesterol-related conditions by about 5% to about 95%.
  • the amount of phospholipase inhibitor used could be the same as the recommended dosage or higher than this dose or lower than the recommended dose.
  • the recommended dosage of a phospholipase inhibitor is between about 0.1 mg/kg/day and about 1,000 mgkg/day.
  • the effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating and/or gastrointestinal concentrations that have been found to be effective in animals, e.g. a mouse model as the ones described in the samples below.
  • a person of ordinary skill in the art can determine phospholipase inhibition by measuring the amount of a product of a phospholipase, e.g., lysophosphatidylcholine (LPC), a product of PL A 2 .
  • the amount of LPC can be determined, for example, by measuring small intestine, lymphatic, and/or serum levels post-prandially.
  • Another technique for determining amount of phospholipase inhibition involves taking direct fluid samples from the gastrointestinal tract.
  • a person of ordinary skill in the art would also be able to monitor in a patient the effect of a phospholipase inhibitor ofthe present invention, e.g., by monitoring cholesterol and/or triglyceride serum levels.
  • the PLA2 inliibitors ofthe invention are preferably lumen-localized PLA2 inhibitors.
  • Such phospholipase inhibitors can be adapted for having both lumen-localization functionality as well as enzyme-inhibition functionalization.
  • certain aspects of such dual functionality can be achieved synergistically (e.g., by using the same structural features and/or charge features); in other schema, the lumen-localization functionality can be achieved independently (e.g., using different structural and/or charge features) from the enzyme-inhibition functionality.
  • Bioavailabihty of this compound can be reduced, and reciprocally, lumen-localization can be improved, according to this prefe ⁇ ed embodiment ofthe invention, for example, by charge modification and/or by covalently linking this indole moiety to a polymer.
  • the phospholipase inhibitors of the invention are preferably localized in the gastrointestinal lumen, such that upon administration to a subject, the phospholipase inhibitors remain substantially in the gastrointestinal lumen. Following administration, the localized phospholipase inhibitors can remain in and pass naturally through the gastrointestinal tract, including the stomach, the duodenum, the small intestine and the large intestine (until passed out ofthe body via the gastrointestinal tract).
  • the phospholipase inhibitors are preferably substantially stable (e.g., with respect to composition and/or with respect to functionality for inhibiting phospholipase) while passing through at least the stomach and the duodenum, and more preferably, are substantially stable while passing through the stomach, the duodenum and the small intestine ofthe gastrointestinal tract, and most preferably, are substantially stable while passing through the entire gastrointestinal tract.
  • the phospholipase inhibitors can act in the gastrointestinal lumen, for example to catabolize phospholipase substrates or to modulate the absorption and/or downstream activities of products of phospholipase digestion.
  • Phospholipase inhibitors are localized within the gastrointestinal lumen, in one approach, by being not absorbed through a gastrointestinal mucosa. As another approach, the phospholipase inhibitors can be localized in the gastrointestinal lumen by being absorbed into a mucosal cell and then effluxed back into a gastrointestinal lumen.
  • the phospholipase inhibitor can be lumen-localized, prefe ⁇ ed phospholipase inhibitors ofthe invention (as contemplated in the various aspects ofthe invention) can be realized by several general lumen- localization embodiments, hi one general lumen-localization embodiment, for example, the phospholipase inhibitor can comprise an oligomer or polymer moiety covalently linked, directly or indirectly through a linking moiety, to a phospholipase inhibiting moiety ofthe invention - including the afore-described indole-related compounds and indole-compounds described herein.
  • the lumen- localized phospholipase inhibitor can be a substituted small organic molecule itself - including the indole-related compounds and indole-compounds described above.
  • the inhibitor can be localized, upon administration to a subject, in the gastrointestinal lumen ofthe subject, such as an animal, and preferably a mammal, including for example a human as well as other mammals (e.g., mice, rats, rabbits, guinea pigs, hamsters, cats, dogs, porcine, poultry, bovine and horses).
  • gastrointestinal lumen is used interchangeably herein with the term "lumen,” to refer to the space or cavity within a gastrointestinal tract, which can also be refe ⁇ ed to as the gut ofthe animal.
  • the phospholipase inhibitor is not absorbed through a gastrointestinal mucosa.
  • Gastrointestinal mucosa refers to the layer(s) of cells separating the gastrointestinal lumen from the rest ofthe body and includes gastric and intestinal mucosa, such as the mucosa ofthe small intestine. In some embodiments, lumen localization is achieved by efflux into the gastrointestinal lumen upon uptake ofthe inhibitor by a gastrointestinal mucosal cell.
  • a "gastrointestinal mucosal cell” as used herein refers to any cell ofthe gastrointestinal mucosa, including, for example, an epithelial cell ofthe gut, such as an intestinal enterocyte, a colonic enterocyte, an apical enterocyte, and the like.
  • the phosphate inhibitor can be an inhibitor that is substantially not absorbed from the gastrointestinal lumen into gastrointestinal mucosal cells.
  • not absorbed as used herein can refer to inhibitors adapted such that a significant amount, preferably a statistically significant amount, more preferably essentially all ofthe phospholipase inhibitor, remains in the gastrointestinal lumen.
  • phospholipase inhibitor For example, at least about 80% of phospholipase inhibitor remains in the gastrointestinal lumen, at least about 85% of phospholipase inhibitor remains in the gastrointestinal lumen, at least about 90% of phospholipase inhibitor remains in the gastrointestinal lumen, at least about 95%, at least about 98%, preferably at least about 99%, and more preferably at least about 99.5% remains in the gastrointestinal lumen.
  • a physiologically insignificant amount ofthe phospholipase inhibitor is absorbed into the blood serum ofthe subject following administration to a subject.
  • the phospholipase inhibitor upon administration ofthe phospholipase inhibitor to a subject, not more than about 20% ofthe administered amount of phospholipase inhibitor is in the serum ofthe subject (e.g., based on detectable serum bioavailabihty following administration), preferably not more than about 15% of phospholipase inhibitor, and most preferably not more than about 10% of phospholipase inhibitor is in the serum ofthe subject. In some embodiments, not more than about 5%, not more than about 2%, preferably not more than about 1%, and more preferably not more than about 0.5% is in the serum ofthe subject.
  • localization to the gastrointestinal lumen can refer to reducing net movement across a gastrointestinal mucosa, for example, by way of both transcellular and paracellular transport, as well as by active and/or passive transport.
  • the phospholipase inhibitor in such embodiments is hindered from net permeation of a gastrointestinal mucosal cell in transcellular transport, for example, through an apical cell ofthe small intestine; the phospholipase inhibitor in these embodiments is also hindered from net permeation through the "tight junctions" in paracellular transport between gastrointestinal mucosal cells lining the lumen.
  • the term “not absorbed” is used interchangeably herein with the terms "non-absorbed,” “non-absorbedness,” “non-absorption” and its other grammatical variations.
  • an inhibitor or inhibiting moiety can be adapted to be non-absorbed by modifying the charge and/or size, particularly, as well as additionally other physical or chemical parameters ofthe phospholipase inhibitor.
  • the phospholipase inhibitor is constructed to have a molecular structure that minimizes or nullifies absorption through a gastrointestinal mucosa.
  • the absorption character of a drug can be selected by applying principles of pha ⁇ nacodynamics, for example, by applying Lipinsky's rule, also known as "the rule of five.”
  • Lipinsky shows that small molecule drugs with (i) molecular weight, (ii) number of hydrogen bond donors, (iii) number of hydrogen bond acceptors, and (iv) water/octanol partition coefficient (Moriguchi logP) each greater than a certain threshold value generally do not show significant systemic concentration.
  • non-absorbed phospholipase inhibitors can be constructed to have molecule structures exceeding one or more of Lipinsky's threshold values, and preferably two or more, or three or more, or four or more or each of
  • Lipinsky's threshold values See also Lipinski et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Delivery Reviews, 46:3-26 (2001); and Lipinski, Drug-like properties and the causes o poor solubility and poor permeability, J. Pharm. & Toxicol. Methods, 44:235-249 (2000), incorporated herein by reference.
  • a phospholipase inhibitor ofthe present invention can be constructed to feature one or more ofthe following characteristics: (i) having a MW greater than about 500 Da; (ii) having a total number of NH and/or OH and/or other potential hydrogen bond donors greater than about 5; (iii) having a total number of O atoms and/or N atoms and/or other potential hydrogen bond acceptors greater than about 10; and/or (iv) having a Moriguchi partition coefficient greater than about 10 5 , i.e., logP greater than about 5.
  • Any art known phospholipase inhibitors and/or any phospholipase inhibiting moieties described below can be used in constructing a non- absorbed molecular structure.
  • permeability coefficient can be determined by methods known to those of skill in the art, including for example by Caco-2 cell permeability assay.
  • the human colon adenocarcinoma cell line, Caco-2 can be used to model intestinal drug absorption and to rank compounds based on their permeability. It has been shown, for example, that the apparent permeability values measured in Caco-2 monolayers in the range of lX10 "7 cm/sec or less typically co ⁇ elate with poor human absorption (Artursson P, K. J. (1991). Permeability can also be determined using an artificial membrane as a model of a gastrointestinal mucosa.
  • a synthetic membrane can be impregnated with e.g. lecithin and/or dodecane to mimic the net permeability characteristics of a gastrointestinal mucosa.
  • the membrane can be used to separate a compartment containing the phospholipase inhibitor from a compartment where the rate of permeation will be monitored.
  • PAMPA parallel artificial membrane permeability assays
  • the phospholipase inhibitor permeability coefficient Log Pe is preferably lower than about -4, or lower than about -4.5, or lower than about -5, more preferably lower than about -5.5, and even more preferably lower than about -6 when measured in the permeability experiment described in Washsland et al. J.Med. Chem. 2001, 44. 923-930.
  • a phospholipase inhibitor can comprise a phospholipase inhibiting moiety such as the indole-related compounds and indole compounds described above, that are linked, coupled or otherwise attached to a non-absorbed oligomer or polymer moiety, where such oligomer or polymer moiety can be a hydrophobic moiety, hydrophilic moiety, and/or charged moiety.
  • the phospholipase inhibiting moiety is coupled to a polymer moiety.
  • such polymer inhibitor can be sized to be non-absorbed, and can be adapted to be enzyme-inhibiting, for example based on one or more or a combination of features, such as charge characteristics, relative balance and/or distribution of hydrophilic / hydrophobic character, and molecular structure.
  • the oligomer or polymer in this general embodiment is preferably soluble, and can preferably be a copolymer (including polymers having two monomer- repeat-units, terpolymers and higher-order polymers), including for example random copolymer or block copolymer.
  • the oligomer or polymer can generally include one or more ionic monomer moieties such as one or more anionic monomer moieties.
  • the oligomer or polymer can generally include one or more hydrophobic monomer moieties.
  • the polymer moiety may be of relatively high molecular weight, for example ranging from about 1000 Da to about 500,000 Da, preferably in the range of about 5000 to about 200,000 Da, and more preferably sufficiently high to hinder or preclude (net) absorption through a gastrointestinal mucosa.
  • Large polymer moieties may be advantageous, for example, in scavenging approaches involving relatively large, soluble or insoluble (e.g., cross-linked) polymers having multiple inhibiting moieties (e.g., as discussed below in connection with Figure 2).
  • the oligomer or polymer moiety may be of low molecular weight, for example not more than about 5000 Da, and preferably not more than about 3000 Da and in some cases not more than about 1000 Da.
  • the oligomer or polymer moiety can consist essentially of or can comprise a block of hydrophobic polymer, allowing the inhibitor to associate with a water-lipid interface.
  • a phospholipase inhibitor for example a composition comprising a phospholipase inhibiting moiety disclosed herein, can be used in a mouse model to demonstrate, for example, suppression of diet-induced insulin resistance, relating to, for example, diet-induced onset of diabetes.
  • the phospholipase inhibitor can be administered to subject animals either as a chow supplement and/or by oral gavage BID in a certain dosage (e.g., less than about 1 ml/kg body weight, or about 25 to about 50 ⁇ l/dose).
  • a typical vehicle for inhibitor suspension comprises about 0.9% carboxymethylcellulose, about 9% PEG-400, and about 0.05% Tween 80, with an inhibitor concentration of about 5 to about 13 mg/ml.
  • This suspension can be added as a supplement to daily chow, e.g., less than about 0.015% ofthe diet by weight, and/or administered by oral gavage BID, e.g., with a daily dose of about 10 mg/kg to about 90 mg/kg body weight.
  • the mouse chow used may have a composition representative of a
  • the chow may contain about 21% milk fat and about 0.15% cholesterol by weight in a diet where 42% of total calories are derived from fat. See, e.g., Harlan Teklad, diet TD88137.
  • the vehicle either with or without the inhibitor, can be mixed with the chow and fed to the mice every day for the duration ofthe study.
  • the duration ofthe study is typically about 6 to about 8 weeks, with the subject animals being dosed every day during this period.
  • Typical dosing groups containing about 6 to about 8 animals per group, can be composed of an untreated control group, a vehicle control group, and dose-treated groups ranging from about 10 mg/kg body weight to about 90 mg/kg body weight.
  • an oral glucose tolerance test and/or an insulin sensitivity test can be conducted as follows: [00148] Oral glucose tolerance test - after an overnight fast, mice from each dosing group can be fed a glucose bolus (e.g., by stomach gavage using about 2 g/kg body weight) in about 50 ⁇ l of saline. Blood samples can be obtained from the tail vein before, and about 15, about 30, about 60, and about 120 minutes after glucose administration; blood glucose levels at the various time points can then be determined.
  • a glucose bolus e.g., by stomach gavage using about 2 g/kg body weight
  • mice in each ofthe dosing groups can be administered bovine insulin (e.g., about lU/kg body weight, using, e.g., intraperitoneal administration.
  • Blood samples can be obtained from the tail vein before, and about 15, about 30, about 60, and about 120 minutes after insulin administration; plasma insulin levels at the various time points can then be determined, e.g., by radioimmunoassay.
  • the effect ofthe non-absorbed phospholipase inhibitor is a decrease in insulin resistance, i.e., better tolerance to glucose challenge by, for example, increasing the efficiency of glucose metabolism in cells, and in the animals ofthe dose-treated groups fed a Western (high fat/high cholesterol) diet relative to the animals ofthe control groups. Effective dosages can also be determined.
  • Example 2 Reduction in fat absorption in a mouse model
  • a phospholipase inhibitor for example a composition comprising a phospholipase inhibiting moiety disclosed herein, can be used in a mouse model to demonstrate, for example, reduced lipid absorption in subjects on a Western diet.
  • the phospholipase inhibitor can be administered to subject animals either as a chow supplement and/or by oral gavage BID in a certain dosage (e.g., less than about 1 ml/kg body weight, or about 25 to about 50 ⁇ l/dose).
  • a typical vehicle for inhibitor suspension comprises about 0.9% carboxymethylcellulose, about 9% PEG-400, and about 0.05% Tween 80, with an inhibitor concentration of about 5 to about 13 mg/ml.
  • This suspension can be added as a supplement to daily chow, e.g., less than about 0.015% ofthe diet by weight, and/or administered by oral gavage BID, e.g., with a daily dose of about 10 mg/kg to 90 mg/kg body weight.
  • the mouse chow used may have a composition representative of a
  • the chow may contain about 21% milk fat and about 0.15% cholesterol by weight in a diet where 42% of total calories are derived from fat. See, e.g., Harlan Teklad, diet TD88137.
  • the vehicle either with or without the inhibitor, can be mixed with the chow and fed to the mice every day for the duration ofthe study.
  • Triglyceride measurements can be taken for a duration of about 6 to about 8 weeks, with the subject animals being dosed every day during this period.
  • Typical dosing groups containing about 6 to about 8 animals per group, can be composed of an untreated control group, a vehicle control group, and dose-treated groups ranging from about 10 mg/kg body weight to about 90 mg/kg body weight.
  • plasma samples can be obtained from the subject animals and analyzed for total triglycerides, for example, to determine the amount of lipids absorbed into the blood circulation.
  • the effect ofthe non-absorbed phospholipase inhibitor e.g., a phospholipase A2 inhibitor, is a net decrease in lipid plasma levels, which indicates reduced fat absorption, in the animals ofthe dose-treated groups fed a Western (high fat/high cholesterol) diet relative to the animals ofthe control groups.
  • Example 3 Reduction in diet-induced hypercholesterolemia in a mouse model
  • a phospholipase inhibitor for example a composition comprising a phospholipase inhibiting moiety disclosed herein, can be used in a mouse model to demonstrate, for example, suppression of diet-induced hypercholesterolemia.
  • the phospholipase inhibitor can be administered to subject animals either as a chow supplement and/or by oral gavage BID (e.g., less than about 1 ml/kg body weight, or about 25 to about 50 ⁇ l/dose).
  • a typical vehicle for inhibitor suspension comprises about 0.9% carboxymethylcellulose, about 9% PEG-400, and about 0.05% Tween 80, with an inhibitor concentration of about 5 to about 13 mg/ml.
  • This suspension can be added as a supplement to daily chow, e.g., less than about 0.015% ofthe diet by weight, and/or administered by oral gavage BID, e.g., with a daily dose of about lOmg/kg to about 90 mg/kg body weight.
  • the mouse chow used may have a composition representative of a
  • the chow may contain about 21% milk fat and about 0.15% cholesterol by weight in a diet where 42% of total calories are derived from fat. See, e.g., Harlan Teklad, diet TD88137.
  • the vehicle either with or without the inhibitor, can be mixed with the chow and fed to the mice every day for the duration ofthe study.
  • Cholesterol and/or triglyceride measurements can be taken for a duration of about 6 to about 8 weeks, with the subject animals being dosed every day during this period.
  • Typical dosing groups containing about 6 to about 8 animals per group, can be composed of a untreated control group, a vehicle control group, and dose-treated groups ranging from about 10 mg/kg body weight to about 90 mg/kg body weight.
  • plasma samples can be obtained from the subject animals and analyzed for total cholesterol and/or triglycerides, for example, to determine the amount of cholesterol and/or lipids absorbed into the blood circulation. Since most plasma cholesterol in a mouse is associated with HDL (in contrast to the LDL association of most cholesterol in humans), HDL and non-HDL fractions can be separated to aid determination ofthe effectiveness ofthe non-absorbed phospholipase inhibitor in lowering plasma non-HDL levels, for example VLDL/LDL.
  • the effect ofthe non-absorbed phospholipase inhibitor is a net decrease in hypercholesterolemia in the animals of the dose-treated groups fed a Western (high fat/high cholesterol) diet relative to the animals ofthe control groups. Effective dosages can also be determined.
  • Example 4 Synthesis ofILY-4001 [2-(3-(2-amino-2-oxoacetyl)-l-(biphenyl-2- ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] (Me Indoxam).
  • This example synthesized a compound for use as a phospholipase inhibitor or inhibiting moiety.
  • N-tert-Butyloxycarbonyl-2-methyl-3-metho ⁇ yaniline (3 [04-035-12].
  • a sti ⁇ ed solution of amine 2 (42.58 g, 0.31 mol) and di-tert-butyl dicarbonate (65.48 g, 0.30 mol) in THF (300 mL) was heated to maintain reflux for 4 h. After cooling to RT, the reaction mixture was concentrated under reduced pressure and the residue was dissolved in EtOAc (500 mL).
  • the reaction mixture was sti ⁇ ed for 1 h at - 60°C and was allowed to warm up to 15°C during 1 h. After cooling to -15°C, the reaction was quenched with 2 ⁇ ⁇ C1 (245 mL) and the resultant mixture was adjusted to p ⁇ of ca. 7 with 2N ⁇ C1. The organic phase was separated off and saved. The aqueous phase was extracted with EtOAc (3 100 mL). The organic solution was concentrated under reduced pressure and the residual pale oil was dissolved in EtOAc (300 mL) and combined with the EtOAc extracts.
  • ILY-4001 the compound 2-(3-(2-amino-2-oxoacetyl)-l-(biphenyl-2-ylmethyl)-2-methyl- lH-indol-4-yloxy)acetic acid is designated as ILY-4001 (and is alternatively refe ⁇ ed to herein as methyl indoxam).
  • ILY-4001 (Fig. 2) was evaluated as a PLA2 IB inhibitor in a set of experiments using wild-type mice and genetically deficient PLA2 (-/-) mice (also refe ⁇ ed to herein as PLA2 knock-out (KO) mice), hi these experiments, wild-type and PLA2 (-/-) mice were maintained on a high fat/high sucrose diet, details of which are described below.
  • ILY-4001 has a measured IC50 value of around 0.2 uM versus the human PLA2 IB enzyme and 0.15 uM versus the mouse PLA2 IB enzyme, in the context ofthe l-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol assay, which measures pyrene substrate release from vesicles treated with PLA2 IB enzyme (Singer, Ghomashchi et al. 2002).
  • An IC-50 value of around 0.062 was determined in experimental studies. (See Example 6A). In addition to its activity against mouse and human pancreatic PLA2, methyl indoxam is stable at low pH, and as such, would be predicted to survive passage through the stomach.
  • ILY-4001 has relatively low absorbtion from the GI lumen, based on Caco-2 assays (See Example 6B), and based on pharmokinetic studies (See Example 6C). [00174] hi the study of this Example 5, twenty-four mice were studied using freatment groups as shown in Table 4, below. Briefly, four groups were set up, each having six mice. Three ofthe groups included six wild-type PLA2 (+/+) mice in each group (eighteen mice total), and one ofthe groups included six genetically deficient PLA2 (-/-) mice. One ofthe wild-type groups was used as a wild-type control group, and was not treated with ILY-4001.
  • the other two wild-type groups were treated with ILY-4001 - one group at a lower dose (indicated as "L” in Table 1) of 25 mg/kg/day, and the other at a higher dose (indicated as "H” in Table 1) of 90 mg/kg/day.
  • the group comprising the PLA2 (-/-) mice was used as a positive confrol group.
  • mice were acclimated for three days on a low fat/low carbohydrate diet. After the three day acclimation phase, the animals were fasted overnight and serum samples taken to establish baseline plasma cholesterol, triglyceride, and glucose levels, along with baseline body weight. The mice in each ofthe freatment groups were then fed a high fat/high sucrose diabetogenic diet (Research Diets D12331).
  • lOOOg ofthe high fat/high sucrose D12331 diet was composed of casein (228g), DL-methionine (2g), maltodextrin 10 (170g), sucrose (175g), soybean oil (25g), hydrogenated coconut oil (333.5g), mineral mix S10001 (40g), sodium bicarbonate (10.5g), potassium citrate (4g), vitamin mix V10001 (lOg), and choline bitartrate (2g).
  • This diet was supplemented with ILY- 4001 treatments such that the average daily dose ofthe compound ingested by a 25g mouse was: 0 mg/kg/day (wild-type control group and PLA2 (-/-) confrol group); 25 mg/kg/day (low-dose wild-type treatment group), or 90 mg/kg/day (high-dose wild-type freatment group).
  • the animals were maintained on the high fat/high sucrose diet, with the designated ILY-4001 supplementation, for a period often weeks.
  • Body weight measurements were taken for all animals in all treatment and control groups at the beginning ofthe treatment period and at 4 weeks and 10 weeks after initiation ofthe study. (See Example 5 A).
  • Example 5A Body-Weight Gain in In-Vivo Evaluation of ILY-4001 [2-(3-(2-amino-2- oxoacetyl)-l-(biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] as PLA2-IB Inhibitor
  • body weight gain for the PLA2 (-/-) mice did not show statistically significant changes from week 4 to week 10, and only a marginal increase in body weight over the extent ofthe study ( ⁇ 5g).
  • the two treatment groups (25 mg/kg/d and 90 mg/kg/d) showed significantly reduced body weight gains at week 4 and week 10 ofthe study compared to the wild- type control group. Both treatment groups showed body weight gain at four weeks modulated to an extent approaching that achieved in the PLA2 (-/-) mice.
  • the low-dose treatment group showed body weight gain at ten weeks modulated to an extent comparable to that achieved in the PLA2 (-/-) mice.
  • Example 5B Fasting Serum Glucose in In-Vivo Evaluation of ILY-4001 [2-(3-(2- amino-2-oxoacetyl)-l-(biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] as PLA2-IB Inhibitor
  • the PLA2 (-/-) KO mice showed a statistically significant decrease in fasting glucose levels at both week 4 and week 10, reflecting an increased sensitivity to insulin not normally seen in mice placed on this diabetogenic diet.
  • the high dose ILY-4001 treatment group (group 3) showed a similar reduction in fasting glucose levels at both four weeks and ten weeks, indicating an improvement in insulin sensitivity for this group as compared to wild-type mice on the high fat/high sucrose diet, and approaching the phenotype seen in the PLA2 (-/-) KO mice.
  • the low dose ILY-4001 treatment group group 2
  • a moderately beneficial effect was seen at week four; however, a beneficial effect was not observed at week ten.
  • Example 5C Serum Cholesterol and Triglycerides in In-Vivo Evaluation of ILY-4001
  • the PLA2 (-/-) KO animals did not show the same increase in these lipids, with cholesterol and triglyceride values each 2 to 3 times lower than those found in the wild-type control group.
  • treatment with ILY-4001 at both the low and high doses substantially reduced the plasma levels of cholesterol and triglycerides, mimicking the beneficial effects at levels comparable to the PLA2 (-/-) KO mice.
  • methyl indoxam (biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid], alternatively refe ⁇ ed to herein as methyl indoxam, with respect to activity, as determined by IC50 assay (Example 6A), with respect to cell absorbtion, as determined by in-vitro Caco-2 assay (Example 6B) and with respect to bioavailabihty, as determined using in-vivo mice studies (Example 6C).
  • Example 6A ICSO Study - ILY-4001 [2-(3-(2-amino-2-oxoacetyl)-l-(biphenyl-2- ylmethyl)-2-methyl-lH-indol-4-yloxy) acetic acid]
  • This example evaluated the IC50 activity value of ILY-4001 [2-(3-(2- amino-2-oxoacetyl)- 1 -(biphenyl-2-ylmethyl)-2-methyl- lH-indol-4-yloxy)acetic acid] , alternatively refe ⁇ ed to herein as methyl indoxam.
  • this assay used a phosphatidylglycerol (or phosphatidylmethanol) substrate with a pyrene fluorophore on the terminal end ofthe sn-2 fatty acyl chain.
  • a phosphatidylglycerol (or phosphatidylmethanol) substrate with a pyrene fluorophore on the terminal end ofthe sn-2 fatty acyl chain.
  • Bovine serum albumin was present in the aqueous phase and captured the pyrene fatty acid when it is liberated from the glycerol backbone owing to the PLA2-catalyzed reaction.
  • a potent inhibitor can inhibit the liberation of pyrene fatty acid from the glycerol backbone.
  • Such features allow for a sensitive PLA2 inhibition assay by monitoring the fluorescence of albumin-bound pyrene fatty acid, as represented in Scheme 1 shown in Figure 7 A. The effect of a given inhibitor and inhibitor concentration on any given phospholipase can be determined. [00187] hi this example, the following reagents and equipment were obtained from commercial vendors: 1. Porcine PLA2 IB 2.
  • PPyrPG or PPyrPM stock solution (1 mg/ml) in toluene:isopropanol (1 : 1)
  • Inhibitor stock solution 10 mM
  • BSA bovine serum albumin
  • Stock buffer 50 mM Tris-HCl, pH 8.0, 50 mM KCl and 1 mM CaCl 2
  • Solution C was prepared by adding 30 ul PPyrPG stock solution to 90 ul ethanol, and then all 120 ul of PPyrPG solution was transfe ⁇ ed drop-wise over approximately 1 min to the continuously stirring 8.82 ml assay buffer to form a final concentration of 4.2 uM PPyrPG vesicle solution. 5.
  • the SPECTRAmax microplate spectrofluorometer was set at 37°C. 6. 100 ul of solution A was added to each inhibition assay well of a costar 96 well black wall/clear bottom plate 7. 100 ul of solution B was added to each inhibition assay well of a costar 96 well black wall/clear bottom plate. 8.
  • the IC50 was calculated using the BioDataFit 1.02 (Four
  • ⁇ is the value ofthe upper asymptote
  • ⁇ is the value ofthe lower asymptote
  • K is a scaling factor
  • ⁇ is a factor that locates the x-ordinate ofthe point of inflection at
  • ILY4001 resulting in 50% maximal PLA2 activity was calculated to be 0.062uM.
  • Example 6B Caco-2 Absorbtion Study - ILY-4001 [2-(3-(2-amino-2-oxoacetyl)-l-
  • This example evaluated the intestinal absorption of ILY-4001 [2-(3-(2- amino-2-oxoacetyl)-l-(biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid], alternatively refe ⁇ ed to herein as methyl indoxam using in-vitro assays with Caco-2 cells.
  • Monolayers were grown and differentiated in MEM (Mediatech) supplemented with 20% FBS, lOOU/ml penicillin, and lOOug/ml streptomycin at 37°C, 95% humidity, 95% air, and 5% CO 2 .
  • the culture medium was refreshed every 48 hours.
  • the cells were washed in transport buffer made up of HBSS with HEPES and the monolayer integrity was evaluated by measuring the trans-epithelial electrical resistance (TEER) of each well.
  • ILY-4001 and Propranolol were diluted to 50 ug/ml in transport buffer and added to the apical wells separately.
  • 150 ul samples were collected for LC/MS analysis from the basolateral well at 15min, 30min, 45min, lhr, 3hr,and 6hr time points; replacing the volume with pre-warmed fransport buffer after each sampling.
  • the apparent permeabilities in cm/s were calculated based on the equation:
  • dQ/dt is the permeability rate co ⁇ ected for the sampling volumes over time
  • Co is the initial concenfration
  • A is the surface area ofthe monolayer (0.32cm 2 ).
  • TEER measurements were retaken and wells with readings below 350 ohm-cm 2 indicated diminished monolayer integrity such that the data from these wells were not valid for analysis.
  • wells were washed with transport buffer and lOOuM of Lucifer Yellow was added to the apical wells. 15min, 30min, and 45min time points were sampled and analyzed by LC/MS to determine paracellular transport.
  • FIG 8 A in which the apparent permeability (cm/s) for ILY-4001 was determined to be around 1.66 x 10 "7 .
  • Example 6C Pharmokinetic Study - ILY-4001 [2-(3-(2-amino-2-oxoacetyl)-l- (biphenyl-2-ylmethyl)-2-methyl-lH-indol-4-yloxy)acetic acid] (Methyl Indoxam). [00197] This example evaluated the bioavailabihty of ILY-4001 [2-(3-(2-amino-
  • Bioavailabihty was calculated as a ratio of AUC-oral / AUC-intravenous
  • a first set of subject animals were given a measured intravenous (IV) dose of ILY-4001 , followed by a determination of ILY-4001 levels in the blood at various time points after administration (e.g., 5 minutes through 24 hours).
  • Another second set of animals was similarly dosed using oral administration, with blood levels of ILY-4001 determined at various time points after administration (e.g., 30 minutes through 24 hours).
  • the level of ILY-4001 in systemic circulation were determined by generally accepted methods (for example as described in Evans, G., A Handbook of Bioanalysis and Drug Metabolism. Boca Raton, CRC Press (2004)).
  • liquid scintillation/mass specfrometry/mass spectrometry analytical methods were used to quantitate plasma concentrations of ILY-4001 after oral and intravenous administration.
  • Pharmacokinetic parameters that were measured include C ma ⁇ , AUC, t max , t. /2 , and F (bioavailabihty).
  • ILY-4001 was dosed at 3 mg/kg IV and 30 mg/kg oral.

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Abstract

La présente invention fournit des procédés et des compositions pour le traitement de conditions relatives au phospholipase. En particulier, l'invention prévoit un procédé de traitement relatif à l'insuline, des conditions relatives au poids et des conditions relatives au cholestérol chez un sujet animal. Le procédé implique généralement l'administration d'un inhibiteur phospholipase A2 comprenant un composé organique substitué ayant une bague fondue à cinq organes et une bague à six organes, telles que des composés contenant des indoles.
EP05779544A 2004-05-03 2005-05-03 Traitement de conditions relatives au régime utilisant des inhibieursphospholipase-a2 comprenant des indoles et des composés relatifs Withdrawn EP1750699A4 (fr)

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US10/838,879 US20050244367A1 (en) 2004-05-03 2004-05-03 Phospholipase inhibitors localized in the gastrointestinal lumen
PCT/US2005/015416 WO2005112646A2 (fr) 2004-05-03 2005-05-03 Traitement de conditions relatives au régime utilisant des inhibieursphospholipase-a2 comprenant des indoles et des composés relatifs

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EP1750699A4 EP1750699A4 (fr) 2008-01-09

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EP05779544A Withdrawn EP1750699A4 (fr) 2004-05-03 2005-05-03 Traitement de conditions relatives au régime utilisant des inhibieursphospholipase-a2 comprenant des indoles et des composés relatifs
EP05779968A Withdrawn EP1750730A4 (fr) 2004-05-03 2005-05-03 Traitement de l'hypercholesterolemie, de l'hypertriglyceridemie et d'etats lies a des troubles cardiovasculaires avec des inhibiteurs de phosphalipase-a2
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US20070292385A1 (en) 2007-12-20
CA2565416A1 (fr) 2005-12-01
EP1750730A2 (fr) 2007-02-14
JP2007538009A (ja) 2007-12-27
WO2005112953A2 (fr) 2005-12-01
JP2007536249A (ja) 2007-12-13
CA2565384A1 (fr) 2005-11-17
US20050244367A1 (en) 2005-11-03
EP1750730A4 (fr) 2008-01-09
WO2005112646A3 (fr) 2006-05-04
WO2005107766A1 (fr) 2005-11-17
US20080021049A1 (en) 2008-01-24
CA2565448A1 (fr) 2005-12-01
WO2005112953A3 (fr) 2006-04-13
EP1747003A4 (fr) 2008-01-09
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JP2007536243A (ja) 2007-12-13
EP1750699A4 (fr) 2008-01-09

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